Methods and compositions for providing tolerance to multiple herbicides

ABSTRACT

Methods and compositions are provided related to improved plants that are tolerant to more than one herbicide. Particularly, the invention provides plants that are tolerant of glyphosate and are tolerant to at least one ALS inhibitor, and methods of use thereof. The glyphosate/ALS inhibitor-tolerant plants comprise a polynucleotide that encodes a polypeptide that confers tolerance to glyphosate and a polynucleotide that encodes an ALS inhibitor-tolerant polypeptide. In specific embodiments, a plant of the invention expresses a GAT polypeptide and an HRA polypeptide. Methods to control weeds, improve plant yield, and increase transformation efficiencies are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/710,854, filed on Aug. 24, 2005, and U.S. Provisional Application No.60/817,011, filed on Jun. 28, 2006, each of which is incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to multiple herbicide tolerances conferred byexpression of a sequence that confers tolerance to glyphosate inconjunction with the expression of at least one other herbicidetolerance gene.

BACKGROUND OF THE INVENTION

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. An ideal treatment would be one which could be applied to anentire field but which would eliminate only the unwanted plants whileleaving the crop plants unharmed. One such treatment system wouldinvolve the use of crop plants which are tolerant to a herbicide so thatwhen the herbicide was sprayed on a field of herbicide-tolerant cropplants, the crop plants would continue to thrive whilenon-herbicide-tolerant weeds were killed or severely damaged. Ideally,such treatment systems would take advantage of varying herbicideproperties so that weed control could provide the best possiblecombination of flexibility and economy. For example, individualherbicides have different longevities in the field, and some herbicidespersist and are effective for a relatively long time after they areapplied to a field while other herbicides are quickly broken down intoother and/or non-active compounds. An ideal treatment system would allowthe use of different herbicides so that growers could tailor the choiceof herbicides for a particular situation.

Crop tolerance to specific herbicides can be conferred by engineeringgenes into crops which encode appropriate herbicide metabolizing enzymesand/or insensitive herbicide targets. In some cases these enzymes, andthe nucleic acids that encode them, originate in a plant. In othercases, they are derived from other organisms, such as microbes. See,e.g., Padgette et al. (1996) “New weed control opportunities:Development of soybeans with a Roundup Ready® gene” and Vasil (1996)“Phosphinothricin-resistant crops,” both in Herbicide-Resistant Crops,ed. Duke (CRC Press, Boca Raton, Fla.) pp. 54-84 and pp. 85-91. Indeed,transgenic plants have been engineered to express a variety of herbicidetolerance genes from a variety of organisms, including a gene encoding achimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochromeP450 oxidoreductase (Shiota et al. (1994) Plant Physiol. 106: 17). Othergenes that confer tolerance to herbicides include acetohydroxy acidsynthase (“AHAS”), mutations in the native sequence have been found toconfer resistance to multiple types of herbicides on plants expressingit and has been introduced into a variety of plants (see, e.g., Hattoriet al. (1995) Mol. Gen. Genet. 246: 419); glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol. 36: 1687);and genes for various phosphotransferases (Datta et al. (1992) PlantMol. Biol. 20: 619).

One herbicide which has been studied extensively isN-phosphonomethylglycine, commonly referred to as glyphosate. Glyphosateis a broad spectrum herbicide that kills both broadleaf and grass-typeplants due to inhibition of the enzyme5-enolpyruvylshikimate-3-phosphate synthase (also referred to as “EPSPsynthase” or “EPSPS”), an enzyme which is part of the biosyntheticpathway for the production of aromatic amino acids, hormones, andvitamins. Glyphosate-resistant transgenic plants have been producedwhich exhibit a commercially viable level of glyphosate resistance dueto the introduction of a modified Agrobacterium CP4 EPSPS. This modifiedenzyme is targeted to the chloroplast where, even in the presence ofglyphosate, it continues to synthesize EPSP from phosphoenolpyruvic acid(“PEP”) and shikimate-3-phosphate. CP4 glyphosate-resistant soybeantransgenic plants are presently in commercial use (e.g., as sold byMonsanto under the name “Roundup Ready®”).

Other herbicides of interest for commercial crop production includeglufosinate (phosphinothricin) and acetolactate synthase (ALS) chemistrysuch as the sulfonylurea herbicides. Glufosinate is a broad spectrumherbicide which acts on the chloroplast glutamate synthase enzyme.Glufosinate-tolerant transgenic plants have been produced which carrythe bar gene from Streptomyces hygroscopicus. The enzyme encoded by thebar gene has N-acetylation activity and modifies and detoxifiesglufosinate. Glufosinate-tolerant plants are presently in commercial use(e.g., as sold by Bayer under the name “Liberty Link®”). Sulfonylureaherbicides inhibit growth of higher plants by blocking acetolactatesynthase (ALS). Plants containing particular mutations in ALS aretolerant to the ALS herbicides including sulfonylureas. Thus, forexample, sulfonylurea herbicides such as Synchrony (a mixture ofchlorimuron-ethyl plus thifensulfuron-methyl) can be used in conjunctionwith ALS herbicide-tolerant plants such as the STS® soybean (Synchronytolerant soybean) variety which contains a trait that enhances thesoybean's natural tolerance to soybean sulfonylurea herbicides.

While a number of herbicide-tolerant crop plants are presentlycommercially available, one issue that has arisen for many commercialherbicides and herbicide/crop combinations is that individual herbicidestypically have incomplete spectrum of activity against common weedspecies. For most individual herbicides which have been in use for sometime, populations of herbicide resistant weed species and biotypes havebecome more prevalent (see, e.g., Tranel and Wright (2002) Weed Science50: 700-712; Owen and Zelaya (2005) Pest Manag Sci. 61: 301-311).Transgenic plants which are resistant to more than one herbicide havebeen described (see, e.g., WO2005/012515). However, improvements inevery aspect of crop production, weed control options, extension ofresidual weed control, and improvement in crop yield are continuously indemand.

Particularly, due to local and regional variation in dominant weedspecies as well as preferred crop species, a continuing need exists forcustomized systems of crop protection and weed management which can beadapted to the needs of a particular region, geography, and/or locality.For example, a continuing need exists for methods of crop protection andweed management which can reduce: the number of herbicide applicationsnecessary to control weeds in a field; the amount of herbicide necessaryto control weeds in a field; the amount of tilling necessary to producea crop; and/or programs which delay or prevent the development and/orappearance of herbicide-resistant weeds. A continuing need exists formethods of crop protection and weed management which allow the targeteduse of particular herbicide combinations.

SUMMARY OF THE INVENTION

Methods and compositions relating to improved plants that are tolerantto more than one herbicide or class or subclass of herbicides areprovided. Compositions include plants that are tolerant to glyphosate aswell as at least one other herbicide or class or subclass of herbicide,as well as, methods of use thereof. Additional compositions compriseplants that comprise a polynucleotide encoding a polypeptide that canconfer tolerance to glyphosate and a polynucleotide encoding an ALSinhibitor-tolerant polypeptide. In one non-limiting embodiment,compositions comprise a plant expressing a polynucleotide encoding a GAT(glyphosate-N-acetyltransferase) polypeptide and are tolerant to atleast one additional herbicide. In some embodiments, a plant of theinvention expresses a GAT polypeptide and an HRA polypeptide.

Methods for controlling weeds in an area of cultivation employing theplants of the invention are provided. Further provided are improvedmethods of transformation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides examples of constructs having 35S enhancer elements.

FIG. 2 provides a schematic demonstrating the effect of 35S enhancers onTX efficiency.

FIG. 3 provides a schematic demonstrating the effect of 35S enhancers onT0 efficiency.

FIG. 4 provides a schematic demonstrating the effect of 35S enhancers onevent copy number.

FIG. 5 provides a table showing the effect of the 35S enhancers on T2efficiency.

FIG. 6 provides an insecticidal gene evaluation assay.

FIG. 7 provides a schematic showing the development of a GAT selectionscheme.

FIG. 8 demonstrates that GAT can be used as a selectable marker.

FIG. 9 provides a schematic demonstrating GAT transformationefficiencies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for making andusing a plant that is tolerant to more than one herbicide or class orsubclass of herbicide. In some embodiments, a plant is provided that istolerant to both glyphosate and at least one other herbicide (or classor subclass of herbicide) or another chemical (or class or subclass ofanother chemical). Such plants find use, for example, in methods ofgrowing crop plants involving treatment with multiple herbicides. Thus,the invention provides improved plants which tolerate treatment with aherbicide or a combination of herbicides (including a combination ofherbicides which act through different modes of action; i.e.,application of mixtures having 2, 3, 4, or more modes of herbicideaction) or a combination of at least one herbicide and at least oneother chemical, including fungicides, insecticides, plant growthregulators and the like. In this manner, the invention provides improvedmethods of growing crop plants in which weeds are selectivelycontrolled. In one embodiment, the plants of the invention comprise apolynucleotide which encodes a polypeptide that confers tolerance toglyphosate and a polynucleotide encoding an ALS inhibitor-tolerantpolypeptide. As discussed in further detail below, such plants arereferred to herein as “glyphosate/ALS inhibitor-tolerant plants.”

The plants of the invention display a modified tolerance to herbicidesand therefore allow for the application of herbicides at rates thatwould significantly damage plants and further allow for the applicationof mixtures of herbicides at lower concentrations than normally appliedbut which still continue to selectively control weeds. In addition, theglyphosate/ALS inhibitor-tolerant plants of the invention can be used incombination with herbicide blends technology and thereby make theapplication of chemical pesticides more convenient, economical, andeffective for the producer. In the case of the glyphosate/ALSinhibitor-tolerant crops, the blends technology will provide easilyformulated crop protection products, of ALS herbicides for example, in adry granule form that enables delivery of customized mixtures designedto solve specific problems in conjunction with the glyphosate/ALSinhibitor-tolerant crop of the invention.

With the addition of robust ALS tolerance afforded by the plants of theinvention, the utility of ALS herbicides is further enabled wherebyherbicidal crop response is eliminated. These uniquely selectiveherbicide offerings coupled with glyphosate/ALS inhibitor tolerate cropsdisclosed herein can now be designed and customized to meetever-changing weed control needs. This breakthrough now enables a myriadof herbicide blends, including for example ALS inhibitor blends, thatcan be customized for improved weed management (since ALS inhibitorchemistries have different herbicidal attributes) including increasedweed spectrum, the ability to provide specified residual activity, asecond mode of action to combat or delay weed resistance (complementingglyphosate, glufosinate or the like), as well as, new offerings that canbe designed either or both as pre-emergence or post-emergence. Blendsalso afford the ability to add or tank mix other agrochemicals atnormal, labeled use rates such as additional herbicides with a 3^(rd) or4^(th) mechanism of action, to fill spectrum holes or even the abilityto include fungicides, insecticides, plant growth regulators and thelike thereby saving costs associated with additional applications. Asdiscussed in further detail below, the methods of the invention can becustomized for a particular location or region. Improved methods oftransformation are also provided.

1. Glyphosate/ALS Inhibitor-Tolerant Plants

a. Glyphosate Tolerance

Plants are provided which comprise a polynucleotide which encodes apolypeptide that confers tolerance to glyphosate and a polynucleotideencoding an ALS inhibitor-tolerant polypeptide. Various sequences whichconfer tolerance to glyphosate can be employed in the methods andcompositions of the invention.

In one embodiment, the mechanism of glyphosate resistance is provided bythe expression of a polynucleotide having transferase activity. As usedherein, a “transferase” polypeptide has the ability to transfer theacetyl group from acetyl CoA to the N of glyphosate, transfer thepropionyl group of propionyl CoA to the N of glyphosate, or to catalyzethe acetylation of glyphosate analogs and/or glyphosate metabolites,e.g., aminomethylphosphonic acid. Methods to assay for this activity aredisclosed, for example, in U.S. Publication No. 2003/0083480, U.S.Publication No. 2004/0082770, and U.S. application Ser. No. 10/835,615,filed Apr. 29,2004, WO2005/012515, WO2002/36782 and WO2003/092360. Inone embodiment, the transferase polypeptide comprises aglyphosate-N-acetyltransferase “GAT” polypeptide.

As used herein, a GAT polypeptide or enzyme comprises a polypeptidewhich has glyphosate-N-acetyltransferase activity (“GAT” activity),i.e., the ability to catalyze the acetylation of glyphosate. In specificembodiments, a polypeptide having glyphosate-N-acetyltransferaseactivity can transfer the acetyl group from acetyl CoA to the N ofglyphosate. In addition, some GAT polypeptides transfer the propionylgroup of propionyl CoA to the N of glyphosate. Some GAT polypeptides arealso capable of catalyzing the acetylation of glyphosate analogs and/orglyphosate metabolites, e.g., aminomethylphosphonic acid. GATpolypeptides are characterized by their structural similarity to oneanother, e.g., in terms of sequence similarity when the GAT polypeptidesare aligned with one another. Exemplary GAT polypeptides and thepolynucleotides encoding them are known in the art and particularlydisclosed, for example, in U.S. application Ser. No. 10/004,357, filedOct. 29, 2001, U.S. application Ser. No. 10/427,692, filed Apr. 30,2003, and U.S. application Ser. No. 10/835,615, filed Apr. 29, 2004,each of which is herein incorporated by reference in its entirety. Insome embodiments, GAT polypeptides used in creating plants of theinvention comprise the amino acid sequence set forth in: SEQ ID NO: 5,14, 11, 8, 21, 27, 17, 24, 30, 35, 46, 47, 48, 49, 50, 51, 52, 53, 39,42, 45, or 54. Each of these sequences is also disclosed in U.S.application Ser. No. 10/835,615, filed Apr. 29, 2004. In someembodiments, the corresponding GAT polynucleotides that encode thesepolypeptides are used; these polynucleotide sequences are set forth inSEQ ID NO: 3, 12, 9, 6, 19, 15, 25, 22, 28, 33, 4, 7, 10, 13, 16, 18,20, 23, 26, 29, 32, 34, 36, 38, 41, 44, 43, 56, 31, 37, 40, 57, 58, 59,60, 61, 62, 63, or 64. Each of these sequences is also disclosed in U.S.application Ser. No. 10/835,615, filed Apr. 29, 2004. As discussed infurther detail elsewhere herein, the use of fragments and variants ofGAT polynucleotides and other known herbicide-tolerance polynucleotidesand polypeptides encoded thereby is also encompassed by the presentinvention.

In specific embodiments, the glyphosate/ALS inhibitor-tolerant plants ofthe invention express a GAT polypeptide, i.e., a polypeptide havingglyphosate-N-acetyltransferase activity wherein the acetyl group fromacetyl CoA is transferred to the N of glyphosate. Thus, plants of theinvention that have been treated with glyphosate contain the glyphosatemetabolite N-acetylglyphosate (“NAG”). Thus, the invention also providesplants that contain NAG as well as a method for producing NAG bytreating plants that contain a GAT gene (i.e., that express a GATpolypeptide) with glyphosate. The presence of N-acetylglyphosate canserve as a diagnostic marker for the presence of an active GAT gene in aplant and can be evaluated by methods known in the art, for example, bymass spectrometry or by immunoassay. Generally, the level of NAG in aplant containing a GAT gene that has been treated with glyphosate iscorrelated with the activity of the GAT gene and the amount ofglyphosate with which the plant has been treated.

The plants of the invention can comprise multiple GAT polynucleotides(i.e., at least 1, 2, 3, 4, 5, 6 or more). It is recognized that ifmultiple GAT polynucleotides are employed, the GAT polynucleotides mayencode GAT polypeptides having different kinetic parameters, i.e., a GATvariant having a lower K_(m) can be combined with one having a higherk_(cat). In some embodiments, the different polynucleotides may becoupled to a chloroplast transit sequence or other signal sequencethereby providing polypeptide expression in different cellularcompartments, organelles or secretion of one or more of thepolypeptides.

The GAT polypeptide encoded by a GAT polynucleotide may have improvedenzymatic activity in comparison to previously identified enzymes.Enzymatic activity can be characterized using the conventional kineticparameters k_(cat), K_(M), and k_(cat)/K_(M). k_(cat) can be thought ofas a measure of the rate of acetylation, particularly at high substrateconcentrations; K_(M) is a measure of the affinity of the GAT enzyme forits substrates (e.g, acetyl CoA, propionyl CoA and glyphosate); andk_(cat)/K_(M) is a measure of catalytic efficiency that takes bothsubstrate affinity and catalytic rate into account. k_(cat)/K_(M) isparticularly important in the situation where the concentration of asubstrate is at least partially rate-limiting. In general, a GAT with ahigher k_(cat) or k_(cat)/K_(M) is a more efficient catalyst thananother GAT with lower k_(cat) or k_(cat)/K_(M). A GAT with a lowerK_(M) is a more efficient catalyst than another GAT with a higher K_(M).Thus, to determine whether one GAT is more effective than another, onecan compare kinetic parameters for the two enzymes. The relativeimportance of k_(cat), k_(cat)/K_(M) and K_(M) will vary depending uponthe context in which the GAT will be expected to function, e.g., theanticipated effective concentration of glyphosate relative to the K_(M)for glyphosate. GAT activity can also be characterized in terms of anyof a number of functional characteristics, including but not limited tostability, susceptibility to inhibition, or activation by othermolecules.

Thus, for example, the GAT polypeptide may have a lower K_(M) forglyphosate than previously identified enzymes, for example, less than 1mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1mM, 0.05 mM, or less. The GAT polypeptide may have a higher k_(cat) forglyphosate than previously identified enzymes, for example, a k_(cat) ofat least 500 min⁻¹, 1000 min⁻¹, 1100 min⁻¹, 1200 min⁻¹, 1250 min⁻¹, 1300min⁻¹, 1400 min⁻¹, 1500 min⁻¹, 1600 min⁻¹, 1700 min¹⁻, 1800 min⁻¹, 1900min⁻¹, or 2000 min⁻¹ or higher. GAT polypeptides for use in theinvention may have a higher k_(cat)/K_(M) for glyphosate than previouslyidentified enzymes, for example, a k_(cat)/K_(M) of at least 1000mM⁻¹min⁻¹, 2000 mM⁻¹min⁻¹, 3000 mM⁻¹min⁻, 4000 mM⁻¹min⁻¹, 5000mM⁻¹min⁻¹, 6000 mM⁻¹min⁻¹, 7000 mM⁻¹min⁻¹, or 8000 mM⁻¹min⁻¹, or higher.The activity of GAT enzymes is affected by, for example, pH and saltconcentration; appropriate assay methods and conditions are known in theart (see, e.g., WO2005012515). Such improved enzymes may find particularuse in methods of growing a crop in a field where the use of aparticular herbicide or combination of herbicides and/or otheragricultural chemicals would result in damage to the plant if theenzymatic activity (i.e., k_(cat), K_(M), or k_(cat)/K_(M)) were lower.

Glyphosate-tolerant plants can also be produced by modifying the plantto increase the capacity to produce a higher level of5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as more fullydescribed in U.S. Pat. Nos. 6,248,876; 5,627,061; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; U.S. Pat. No. Re. 36,449; U.S. Pat. No. RE 37,287 E; and U.S.Pat. No. 5,491,288; and international publications WO 97/04103; WO00/66746; WO 01/66704; and WO 00/66747, which are incorporated herein byreference in their entireties for all purposes. Glyphosate resistancecan also be imparted to plants that express a gene that encodes aglyphosate oxido-reductase enzyme as described more fully in U.S. Pat.Nos. 5,776,760 and 5,463,175, which are incorporated herein by referencein their entireties for all purposes. Additionally, glyphosate tolerantplants can be generated through the selection of naturally occurringmutations that impart tolerance to glyphosate.

It is recognized that the methods and compositions of the invention canemploy any combination of sequences (i.e., sequences that act via thesame or different modes) that confer tolerance to glyphosate known inthe art to produce plants and plant explants with superior glyphosateresistance.

b. Acetolactate Synthase (ALS) Inhibitor Tolerance

Glyphosate/ALS inhibitor-tolerant plants are provided which comprise apolynucleotide which encodes a polypeptide that confers tolerance toglyphosate and further comprise a polynucleotide encoding anacetolactate synthase (ALS) inhibitor-tolerant polypeptide. As usedherein, an “ALS inhibitor-tolerant polypeptide” comprises anypolypeptide which when expressed in a plant confers tolerance to atleast one ALS inhibitor. A variety of ALS inhibitors are known andinclude, for example, sulfonylurea, imidazolinone, triazolopyrimidines,pryimidinyoxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinoneherbicide. Additional ALS inhibitors are known and are disclosedelsewhere herein. It is known in the art that ALS mutations fall intodifferent classes with regard to tolerance to sulfonylureas,imidazolinones, triazolopyrimidines, and pyrimidinyl(thio)benzoates,including mutations having the following characteristics: (1) broadtolerance to all four of these groups; (2) tolerance to imidazolinonesand pyrimidinyl(thio)benzoates; (3) tolerance to sulfonylureas andtriazolopyrimidines; and (4) tolerance to sulfonylureas andimidazolinones.

Various ALS inhibitor-tolerant polypeptides can be employed. In someembodiments, the ALS inhibitor-tolerant polynucleotides contain at leastone nucleotide mutation resulting in one amino acid change in the ALSpolypeptide. In specific embodiments, the change occurs in one of sevensubstantially conserved regions of acetolactate synthase. See, forexample, Hattori et al. (1995) Molecular Genetics and Genomes246:419-425; Lee et al. (1998) EMBO Journal 7:1241-1248; Mazur et al.(1989) Ann. Rev. Plant Phys. 40:441-470; and U.S. Pat. No. 5,605,011,each of which is incorporated by reference in their entirety. The ALSinhibitor-tolerant polypeptide can be encoded by, for example, the SuRAor SuRB locus of ALS. In specific embodiments, the ALSinhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALSmutant, the S4 mutant or the S4/HRA mutant or any combination thereof.Different mutations in ALS are known to confer tolerance to differentherbicides and groups (and/or subgroups) of herbicides; see, e.g.,Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Pat.No. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which isherein incorporated by reference in their entirety. See also, SEQ IDNO:65 comprising a soybean HRA sequence; SEQ ID NO:66 comprising a maizeHRA sequence; SEQ ID NO:67 comprising an Arabidopsis HRA sequence; andSEQ ID NO:86 comprising an HRA sequence used in cotton. The HRA mutationin ALS finds particular use in one embodiment of the invention. Themutation results in the production of an acetolactate synthasepolypeptide which is resistant to at least one ALS inhibitor chemistryin comparison to the wild-type protein. For example, a plant expressingan ALS inhibitor-tolerant polypeptide may be tolerant of a dose ofsulfonylurea, imidazolinone, triazolopyrimidines,pryimidinyloxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinoneherbicide that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 50, 70, 80, 100, 125, 150, 200, 500, or 1000 times higher than adose of the herbicide that would cause damage to an appropriate controlplant. In some embodiments, an ALS inhibitor-tolerant polypeptidecomprises a number of mutations. Additionally, plants having an ALSinhibitor polypeptide can be generated through the selection ofnaturally occurring mutations that impart tolerance to glyphosate.

In some embodiments, the ALS inhibitor-tolerant polypeptide conferstolerance to sulfonylurea and imidazolinone herbicides. Sulfonylurea andimidazolinone herbicides inhibit growth of higher plants by blockingacetolactate synthase (ALS), also known as, acetohydroxy acid synthase(AHAS). For example, plants containing particular mutations in ALS(e.g., the S4 and/or HRA mutations) are tolerant to sulfonylureaherbicides. The production of sulfonylurea-tolerant plants andimidazolinone-tolerant plants is described more fully in U.S. Pat. Nos.5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732;4,761,373; 5,331,107; 5,928,937; and 5,378,824; and internationalpublication WO 96/33270, which are incorporated herein by reference intheir entireties for all purposes. In specific embodiments, the ALSinhibitor-tolerant polypeptide comprises a sulfonamide-tolerantacetolactate synthase (otherwise known as a sulfonamide-tolerantacetohydroxy acid synthase) or an imidazolinone-tolerant acetolactatesynthase (otherwise known as an imidazolinone-tolerant acetohydroxy acidsynthase).

A plant of the invention that comprises at least one sequence whichconfers tolerance to glyphosate and at least one sequence which conferstolerance to an ALS inhibitor is referred to herein as a “glyphosate/ALSinhibitor-tolerant plant.” A plant of the invention that contains atleast one GAT polypeptide and at least one HRA polypeptide is referredto herein as a “GAT-HRA plant.”

c. Additional Herbicide Tolerance

In some embodiments, plants are provided having enhanced tolerance toglyphosate and at least one ALS inhibitor herbicide, as well as,tolerance to at least one additional herbicide. In specific embodiments,tolerance to the additional herbicide is due to the expression of atleast one polypeptide imparting tolerance to the additional herbicide.In some embodiments, a composition of the invention (e.g., a plant) maycomprise two, three, four, five, six, seven, or more traits which confertolerance to at least one herbicide, so that a plant of the inventionmay be tolerant to at least two, three, four, five, six, or seven ormore different types of herbicides. Thus, a plant of the invention thatis tolerant to more than two different herbicides may be tolerant toherbicides that have different modes of action and/or different sites ofaction. In some embodiments, all of these traits are transgenic traits,while in other embodiments, at least one of these traits is nottransgenic.

In some of these embodiments, each herbicide tolerance gene conferstolerance to a different herbicide or class or subclass of herbicides.In some of these embodiments, at least two of the herbicide tolerancegenes confer tolerance to the same herbicide or to members of the sameclass or subclass of herbicides. Accordingly, further provided areplants having a polynucleotide that encodes a polypeptide which canconfer tolerance to glyphosate and a polynucleotide that encodes an ALSinhibitor-tolerant polypeptide can further comprise at least oneadditional herbicide-tolerance polynucleotide which when expressedimparts tolerance to an additional herbicide. Such additionalherbicides, include but are not limited to, an acetyl Co-A carboxylaseinhibitor such as quizalofop-P-ethyl, a synthetic auxin such asquinclorac, a protoporphyrinogen oxidase (PPO) inhibitor herbicide (suchas sulfentrazone), a pigment synthesis inhibitor herbicide such as ahydroxyphenylpyruvate dioxygenase inhibitor (e.g., mesotrione orsulcotrione), a phosphinothricin acetyltransferase or a phytoenedesaturase inhibitor like diflufenican or pigment synthesis inhibitor.It is understood that the invention is not bound by the mechanism ofaction of a herbicide, so long as the goal of the invention (i.e.,herbicide tolerance to glyphosate and at least on ALS inhibitor) isachieved. Additional herbicides of interest are disclosed elsewhereherein.

In some embodiments, the compositions of the invention further comprisepolypeptides conferring tolerance to herbicides which inhibit the enzymeglutamine synthase, such as phosphinothricin or glufosinate (e.g., thebar gene or pat gene). Glutamine synthetase (GS) appears to be anessential enzyme necessary for the development and life of most plantcells, and inhibitors of GS are toxic to plant cells. Glufosinateherbicides have been developed based on the toxic effect due to theinhibition of GS in plants. These herbicides are non-selective; that is,they inhibit growth of all the different species of plants present. Thedevelopment of plants containing an exogenous phosphinothricinacetyltransferase is described in U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616; and 5,879,903, which are incorporated herein by reference intheir entireties for all purposes. Mutated phosphinothricinacetyltransferase having this activity are also disclosed.

In still other embodiments, the compositions of the invention furthercomprise polypeptides conferring tolerance to herbicides which inhibitprotox (protoporphyrinogen oxidase). Protox is necessary for theproduction of chlorophyll, which is necessary for all plant survival.The protox enzyme serves as the target for a variety of herbicidalcompounds. These herbicides also inhibit growth of all the differentspecies of plants present. The development of plants containing alteredprotox activity which are resistant to these herbicides are described inU.S. Pat. Nos. 6,288,306; 6,282,837; and 5,767,373; and internationalpublication WO 01/12825, which are incorporated herein by reference intheir entireties for all purposes.

In still other embodiments, compositions of the invention may comprisepolypeptides involving other modes of herbicide resistance. For example,hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reactionin which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. Molecules which inhibit this enzyme and which bind to theenzyme in order to inhibit transformation of the HPP into homogentisateare useful as herbicides. Plants more resistant to certain herbicidesare described in U.S. Pat. Nos. 6,245,968; 6,268,549; and 6,069,115; andinternational publication WO 99/23886, which are incorporated herein byreference in their entireties for all purposes. Mutatedhydroxyphenylpyruvatedioxygenase having this activity are alsodisclosed.

d. Fragments and Variants of Sequences that Confer Herbicide Tolerance

Depending on the context, “fragment” refers to a portion of thepolynucleotide or a portion of the amino acid sequence and hence proteinencoded thereby. Fragments of a polynucleotide may encode proteinfragments that retain the biological activity of the original proteinand hence confer tolerance to a herbicide. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the full-lengthpolynucleotide encoding a herbicide-tolerance polypeptide.

A fragment of a herbicide-tolerance polynucleotide that encodes abiologically active portion of a herbicide-tolerance polypeptide willencode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthherbicide-tolerance polypeptide. A biologically active portion of aherbicide-tolerance polypeptide can be prepared by isolating a portionof a herbicide-tolerance polynucleotide, expressing the encoded portionof the herbicide-tolerance polypeptide (e.g., by recombinant expressionin vitro), and assessing the activity of the encoded portion of theherbicide-tolerance polypeptide. Polynucleotides that are fragments of aherbicide-tolerance polynucleotide comprise at least 16, 20, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800,900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides, or upto the number of nucleotides present in a full-lengthherbicide-tolerance polynucleotide.

The term “variants” refers to substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally-occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of aherbicide-tolerance polypeptide. Naturally occurring allelic variantssuch as these can be identified with the use of well-known molecularbiology techniques, as, for example, with polymerase chain reaction(PCR) and hybridization techniques. Variant polynucleotides also includesynthetically derived polynucleotides, such as those generated, forexample, by using site-directed mutagenesis or “shuffling.” Generally,variants of a particular polynucleotide have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters as described elsewhere herein.

Variants of a particular polynucleotide (i.e., the referencepolynucleotide) can also be evaluated by comparison of the percentsequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotides ofthe invention is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from a nativeand/or original protein by deletion (so-called truncation) of one ormore amino acids at the N-terminal and/or C-terminal end of the protein;deletion and/or addition of one or more amino acids at one or moreinternal sites in the protein; or substitution of one or more aminoacids at one or more sites in the protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired herbicide-tolerance activity as described herein.Biologically active variants of a herbicide-tolerance polypeptide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a herbicide-tolerancepolypeptide may differ from that polypeptide by as few as 1-15 aminoacid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4,3, 2, or even 1 amino acid residue. Variant herbicide-tolerancepolypeptides, as well as polynucleotides encoding these variants, areknown in the art.

Herbicide-tolerance polypeptides may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants and fragments ofherbicide-tolerance polypeptides can be prepared by mutations in theencoding polynucleotide. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods inEnzymol. 154: 367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) AtlasofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be made. One skilled in the art will appreciate that theactivity of a herbicide-tolerance polypeptide can be evaluated byroutine screening assays. That is, the activity can be evaluated bydetermining whether a transgenic plant has an increased tolerance to aherbicide, for example, as illustrated in working Example 1, or with anin vitro assay, such as the production of N-acetylglyphosphate fromglyphosate by a GAT polypeptide (see, e.g., WO 02/36782).

Variant polynucleotides and polypeptides also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more differentherbicide-tolerance polypeptide coding sequences can be manipulated tocreate a new herbicide-tolerance polypeptide possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. For example, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between a herbicide-tolerance polypeptide and other known genesto obtain a new gene coding for a protein with an improved property ofinterest, such as an increased K_(m) in the case of an enzyme.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751;Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) NatureBiotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347;Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri etal. (1998) Nature 391: 288-291; and U.S. Pat. Nos. 5,605,793 and5,837,458.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et al (1990) J.Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. BLASTsoftware is publicly available on the NCBI website. Alignment may alsobe performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the GCG Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89: 10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to be limited topolynucleotides comprising DNA. Those of ordinary skill in the art willrecognize that polynucleotides can comprise ribonucleotides andcombinations of ribonucleotides and deoxyribonucleotides. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. Thus, polynucleotides alsoencompass all forms of sequences including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like.

e. Herbicide Tolerance

A “herbicide” is a chemical that causes temporary or permanent injury toa plant. Non-limiting examples of herbicides that can be employed in thevarious methods and compositions of the invention are discussed infurther detail elsewhere herein. A herbicide may be incorporated intothe plant, or it may act on the plant without being incorporated intothe plant or its cells. An “active ingredient” is the chemical in aherbicide formulation primarily responsible for its phytotoxicity andwhich is identified as the active ingredient on the product label.Product label information is available from the U.S. EnvironmentalProtection Agency and is updated online at the urloaspub.epa.gov/pestlabl/ppls.own; product label information is alsoavailable online at the url www.cdms.net. The term “acid equivalent”expresses the rate or quantity as the herbicidally active parent acid.For example, 2,4-D acid is often formulated in the form of a sodium oramine salt or an ester as the active ingredient in formulated products.The active acid equivalent per gallon of a widely used ester formulationis 3.8 lb a.e./gallon (about 0.454 kg a.e./L), while the activeingredient per gallon is 6.0 lb ai/gallon (about 0.717 kg ai/L). As usedherein, an “agricultural chemical” is any chemical used in the contextof agriculture.

“Herbicide-tolerant” or “tolerant” or “crop tolerance” in the context ofherbicide or other chemical treatment as used herein means that a plantor other organism treated with a particular herbicide or class orsubclass of herbicide or other chemical or class or subclass of otherchemical will show no significant damage or less damage following thattreatment in comparison to an appropriate control plant. A plant may benaturally tolerant to a particular herbicide or chemical, or a plant maybe herbicide-tolerant as a result of human intervention such as, forexample, breeding or genetic engineering. An “herbicide-tolerancepolypeptide” is a polypeptide that confers herbicide tolerance on aplant or other organism expressing it (i.e., that makes a plant or otherorganism herbicide-tolerant), and an “herbicide-tolerancepolynucleotide” is a polynucleotide that encodes a herbicide-tolerancepolypeptide. For example, a sulfonylurea tolerant polypeptide is onethat confers tolerance to sulfonylurea herbicides on a plant or otherorganism that expresses it, an imidazolinone tolerant polypeptide is onethat confers tolerance to imidazolinone herbicides on a plant or otherorganism that expresses it; and a glyphosate tolerant polypeptide is onethat confers tolerance to glyphosate on a plant or other organism thatexpresses it.

Thus, a plant is tolerant to a herbicide or other chemical if it showsdamage in comparison to an appropriate control plant that is less thanthe damage exhibited by the control plant by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%,150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% ormore. In this manner, a plant that is tolerant to a herbicide or otherchemical shows “improved tolerance” in comparison to an appropriatecontrol plant. Damage resulting from herbicide or other chemicaltreatment is assessed by evaluating any parameter of plant growth orwell-being deemed suitable by one of skill in the art. Damage can beassessed by visual inspection and/or by statistical analysis of suitableparameters of individual plants or of a group of plants. Thus, damagemay be assessed by evaluating, for example, parameters such as plantheight, plant weight, leaf color, leaf length, flowering, fertility,silking, yield, seed production, and the like. Damage may also beassessed by evaluating the time elapsed to a particular stage ofdevelopment (e.g., silking, flowering, or pollen shed) or the timeelapsed until a plant has recovered from treatment with a particularchemical and/or herbicide.

In making such assessments, particular values may be assigned toparticular degrees of damage so that statistical analysis orquantitative comparisons may be made. The use of ranges of values todescribe particular degrees of damage is known in the art, and anysuitable range or scale may be used. For example, herbicide injuryscores (also called tolerance scores) can be assigned as illustrated inExample 1 using the scale set forth in Table 7. In this scale, a ratingof 9 indicates that a herbicide treatment had no effect on a crop, i.e.,that no crop reduction or injury was observed following the herbicidetreatment. Thus, in this scale, a rating of 9 indicates that the cropexhibited no damage from the herbicide and therefore that the crop istolerant to the herbicide. As indicated above, herbicide tolerance isalso indicated by other ratings in this scale where an appropriatecontrol plant exhibits a lower score on the scale, or where a group ofappropriate control plants exhibits a statistically lower score inresponse to a herbicide treatment than a group of subject plants.

Damage caused by a herbicide or other chemical can be assessed atvarious times after a plant has been treated with a herbicide. Often,damage is assessed at about the time that the control plant exhibitsmaximum damage. Sometimes, damage is assessed after a period of time inwhich a control plant that was not treated with herbicide or otherchemical has measurably grown and/or developed in comparison to the sizeor stage at which the treatment was administered. Damage can be assessedat various times, for example, at 12 hours or at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 days, or three weeks, four weeks, or longer afterthe test plant was treated with herbicide. Any time of assessment issuitable as long as it permits detection of a difference in response toa treatment of test and control plants.

A herbicide does not “significantly damage” a plant when it either hasno effect on a plant or when it has some effect on a plant from whichthe plant later recovers, or when it has an effect which is detrimentalbut which is offset, for example, by the impact of the particularherbicide on weeds. Thus, for example, a crop plant is not“significantly damaged by” a herbicide or other treatment if it exhibitsless than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% decrease in at least one suitable parameter that is indicativeof plant health and/or productivity in comparison to an appropriatecontrol plant (e.g., an untreated crop plant). Suitable parameters thatare indicative of plant health and/or productivity include, for example,plant height, plant weight, leaf length, time elapsed to a particularstage of development, flowering, yield, seed production, and the like.The evaluation of a parameter can be by visual inspection and/or bystatistical analysis of any suitable parameter. Comparison may be madeby visual inspection and/or by statistical analysis. Accordingly, a cropplant is not “significantly damaged by” a herbicide or other treatmentif it exhibits a decrease in at least one parameter but that decrease istemporary in nature and the plant recovers fully within 1 week, 2 weeks,3 weeks, 4 weeks, or 6 weeks.

Conversely, a plant is significantly damaged by a herbicide or othertreatment if it exhibits more than a 50%, 60%, 70%, 80%, 90%, 100%,110%, 120%, 150%, 170% decrease in at least one suitable parameter thatis indicative of plant health and/or productivity in comparison to anappropriate control plant (e.g., an untreated weed of the same species).Thus, a plant is significantly damaged if it exhibits a decrease in atleast one parameter and the plant does not recover fully within 1 week,2 weeks, 3 weeks, 4 weeks, or 6 weeks.

Damage resulting from a herbicide or other chemical treatment of a plantcan be assessed by visual inspection by one of skill in the art and canbe evaluated by statistical analysis of suitable parameters. The plantbeing evaluated is referred to as the “test plant.” Typically, anappropriate control plant is one that expresses the sameherbicide-tolerance polypeptide(s) as the plant being evaluated forherbicide tolerance (i.e., the “test plant”) but that has not beentreated with herbicide. For example, in evaluating a herbicide-tolerantplant of the invention that confers tolerance to glyphosate and an ALSinhibitor, an appropriate control plant would be a plant that expresseseach of these sequence but is not treated with the herbicide. In somecircumstances, the control plant is one that that has been subjected tothe same herbicide treatment as the plant being evaluated (i.e., thetest plant) but that does not express the enzyme intended to providetolerance to the herbicide of interest in the test plant. One of skillin the art will be able to design, perform, and evaluate a suitablecontrolled experiment to assess the herbicide tolerance of a plant ofinterest, including the selection of appropriate test plants, controlplants, and treatments.

Thus, as used herein, a “test plant or plant cell” is one in whichgenetic alteration has been effected as to at least one gene ofinterest, or is a plant or plant cell which is descended from a plant orcell so altered and which comprises the alteration. A genetic alterationmay be introduced into the plant by breeding or by transformation.“Genetic alteration” is intended to mean a gene or mutation thereofwhich confers a phenotype on the plant that differs from the phenotypeof a plant that does not contain the genetic alteration.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell, and may be any suitable plant or plant cell. A controlplant or plant cell may comprise, for example: (a) a wild-type plant orcell, i.e., of the same genotype as the starting material for thegenetic alteration which resulted in the subject plant or cell; (b) aplant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell which is genetically identical to thesubject plant or plant cell but which is not exposed to the sametreatment (e.g., herbicide treatment) as the subject plant or plantcell; (e) the subject plant or plant cell itself, under conditions inwhich the gene of interest is not expressed; or (f) the subject plant orplant cell itself, under conditions in which it has not been exposed toa particular treatment such as, for example, a herbicide or combinationof herbicides and/or other chemicals. In some instances, an appropriatecontrol plant or control plant cell may have a different genotype fromthe subject plant or plant cell but may share the herbicide-sensitivecharacteristics of the starting material for the genetic alteration(s)which resulted in the subject plant or cell (see, e.g., Green (1998)Weed Technology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516). In some instances, an appropriate control maize plantcomprises a NK603 event (Nielson et al. (2004) European Food Researchand Technology 219:421-427 and Ridley et al. (2002) Journal ofAgriculture and Food Chemistry 50: 7235-7243), an elite stiff stalkinbred plant, a P3162 plant (Pioneer Hi-Bred International), a 39T66plant (Pioneer Hi-Bred International), or a 34M91 plant (Pioneer Hi-BredInternational). In some instances, an appropriate control soybean plantis a “Jack” soybean plant (Illinois Foundation Seed, Champaign, Ill.).

Plants of the invention express a polypeptide that confers tolerance toglyphosate and at least one other polypeptide that confers tolerance toan ALS inhibitor. A plant of the invention shows at least one improvedproperty relative to an appropriate control plant, such as, for example,improved herbicide tolerance, reduced lodging, increased height, reducedtime to maturity, and improved yield. A plant has an improved propertywhen it exhibits a statistically significant difference from anappropriate control plant wherein that difference is in a direction thatrepresents an improvement over the control plant. For example, a planthas an improved property when it exhibits an increase in yield that isstatistically significant in comparison to a control plant, and/or whenit exhibits a decrease in damage resulting from treatment with aherbicide. Techniques for such assessments are known in the art. Anysuitable statistical analysis may be used, such as, for example, anANOVA (available as a commercial package from SAS Institute, Inc., 100SAS Campus Drive, Cary, N.C.).

f. Plants

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, explants, and plant cells thatare intact in plants or parts of plants such as embryos, pollen, ovules,seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, and the like. Grain is intended tomean the mature seed produced by commercial growers for purposes otherthan growing or reproducing the species. Progeny, variants, and mutantsof the regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introducedpolynucleotides. Thus, the invention provides transgenic seeds producedby the plants of the invention.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays, also referred to herein as “maize”), Brassica spp. (e.g., B.napus, B. rapa, B. juncea), particularly those Brassica species usefulas sources of seed oil (also referred to as “canola”), flax (Linumspp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, fruits,ornamentals (flowers), sugar cane, conifers, Arabidopsis.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Any tree can also be employed. Conifers that may be employed inpracticing the present invention include, for example, pines such asloblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine(Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine(Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock(Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Hardwoodtrees can also be employed including ash, aspen, beech, basswood, birch,black cherry, black walnut, buckeye, American chestnut, cottonwood,dogwood, elm, hackberry, hickory, holly, locust, magnolia, maple, oak,poplar, red alder, redbud, royal paulownia, sassafras, sweetgum,sycamore, tupelo, willow, yellow-poplar.

In specific embodiments, plants of the present invention are crop plants(for example, corn (also referred to as “maize”), alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.).

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Other plants of interest include Turfgrasses such as, for example,turfgrasses from the genus Poa, Agrostis, Festuca, Lolium, and Zoysia.Additional turfgrasses can come from the subfamily Panicoideae.Turfgrasses can further include, but are not limited to, Blue gramma(Bouteloua gracilis (H.B.K.) Lag. Ex Griffiths); Buffalograss (Buchloedactyloids (Nutt.) Engelm.); Slender creeping red fescue (Festuca rubrassp. Litoralis); Red fescue (Festuca rubra); Colonial bentgrass(Agrostis tenuis Sibth.); Creeping bentgrass (Agrostis palustris Huds.);Fairway wheatgrass (Agropyron cristatum (L.) Gaertn.); Hard fescue(Festuca longifolia Thuill.); Kentucky bluegrass (Poa pratensis L.);Perennial ryegrass (Lolium perenne L.); Rough bluegrass (Poa trivialisL.); Sideoats grama (Bouteloua curtipendula Michx. Torr.); Smoothbromegrass (Bromus inermis Leyss.); Tall fescue (Festuca arundinaceaSchreb.); Annual bluegrass (Poa annua L.); Annual ryegrass (Loliummultiflorum Lam.); Redtop (Agrostis alba L.); Japanese lawn grass(Zoysia japonica); bermudagrass (Cynodon dactylon; Cynodon spp. L.C.Rich; Cynodon transvaalensis); Seashore paspalum (Paspalum vaginatumSwartz); Zoysiagrass (Zoysia spp. Willd; Zoysia japonica and Z. matrellavar. matrella); Bahiagrass (Paspalum notatum Flugge); Carpetgrass(Axonopus affinis Chase); Centipedegrass (Eremochloa ophiuroides MunroHack.); Kikuyugrass (Pennisetum clandesinum Hochst Ex Chiov); Browntopbent (Agrostis tenuis also known as A. capillaris); Velvet bent(Agrostis canina); Perennial ryegrass (Lolium perenne); and, St.Augustinegrass (Stenotaphrum secundatum Walt. Kuntze). Additionalgrasses of interest include switchgrass (Panicum virgatum).

g. Stacking of Traits and Additional Traits of Interest

In some embodiments, the polynucleotide conferring the glyphosatetolerance and the ALS inhibitor tolerance in the plants of the inventionare engineered into a molecular stack. In other embodiments, themolecular stack further comprises at least one additional polynucleotidethat confers tolerance to a 3^(rd) herbicide. Such sequences aredisclosed elsewhere in herein. In one embodiment, the sequence conferstolerance to glufosinate, and 2 in a specific embodiment, the sequencecomprises pat.

In other embodiments, the glyphosate/ALS inhibitor-tolerant plants ofthe invention comprise one or more trait of interest, and in morespecific embodiments, the plant is stacked with any combination ofpolynucleotide sequences of interest in order to create plants with adesired combination of traits. A trait, as used herein, refers to thephenotype derived from a particular sequence or groups of sequences. Forexample, herbicide-tolerance polynucleotides may be stacked with anyother polynucleotides encoding polypeptides having pesticidal and/orinsecticidal activity, such as Bacillus thuringiensis toxic proteins(described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; Geiser et al. (1986) Gene 48: 109; Lee et al. (2003) Appl.Environ. Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) ActaCrystallogr. D. Biol. Crystallogr. 57:1101-1109 (Cry3Bb1); and Herman etal. (2004) J. Agric. Food Chem. 52: 2726-2734 (Cry1F)), lectins (VanDamme et al. (1994) Plant Mol. Biol. 24: 825, pentin (described in U.S.Pat. No. 5,981,722), and the like. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.

In some embodiments, herbicide-tolerance polynucleotides of theglyphosate/ALS inhibitor-tolerant plants (i.e., such as plant comprisingGAT and HRA) may be stacked with other herbicide-tolerance traits tocreate a transgenic plant of the invention with further improvedproperties. Other herbicide-tolerance polynucleotides that could be usedin such embodiments include those conferring tolerance to glyphosate orto ALS inhibitors by other modes of action, such as, for example, a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175. Other traits that could becombined with herbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., such as GAT and HRA sequence) includethose derived from polynucleotides that confer on the plant the capacityto produce a higher level of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), for example, as more fully described in U.S. Pat. Nos.6,248,876 B 1; 5,627,061; 5,804,425; 5,633,435; 5,145,783; 4,971,908;5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; U.S. Pat. No. Re.36,449; U.S. Pat. Nos. RE 37,287 E; and 5,491,288; and internationalpublications WO 97/04103; WO 00/66746; WO 01/66704; and WO 00/66747.Other traits that could be combined with herbicide-tolerancepolynucleotides of the glyphosate/ALS inhibitor-tolerant plants (i.e.,such as GAT and HRA sequences) include those conferring tolerance tosulfonylurea and/or imidazolinone, for example, as described more fullyin U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180;5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; andinternational publication WO 96/33270.

In some embodiments, herbicide-tolerance polynucleotides of theglyphosate/ALS inhibitor-tolerant plants (i.e., such as GAT and HRAsequence) may be stacked with, for example,hydroxyphenylpyruvatedioxygenases which are enzymes that catalyze thereaction in which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. Molecules which inhibit this enzyme and which bind to theenzyme in order to inhibit transformation of the HPP into homogentisateare useful as herbicides. Traits conferring tolerance to such herbicidesin plants are described in U.S. Pat. Nos. 6,245,968 B 1; 6,268,549; and6,069,115; and international publication WO 99/23886. Other examples ofsuitable herbicide-tolerance traits that could be stacked withherbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., such GAT and HRA sequences) includearyloxyalkanoate dioxygenase polynucleotides (which reportedly confertolerance to 2,4-D and other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inWO2005/107437) and dicamba-tolerance polynucleotides as described, forexample, in Herman et al. (2005) J. Biol. Chem. 280: 24759-24767.

Other examples of herbicide-tolerance traits that could be combined withherbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., GAT and HRA plants) include thoseconferred by polynucleotides encoding an exogenous phosphinothricinacetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616; and 5,879,903. Plants containing an exogenousphosphinothricin acetyltransferase can exhibit improved tolerance toglufosinate herbicides, which inhibit the enzyme glutamine synthase.Other examples of herbicide-tolerance traits that could be combined withthe herbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., GAT and HRA plants) include thoseconferred by polynucleotides conferring altered protoporphyrinogenoxidase (protox) activity, as described in U.S. Pat. Nos. 6,288,306 B1;6,282,837 B1; and 5,767,373; and international publication WO 01/12825.Plants containing such polynucleotides can exhibit improved tolerance toany of a variety of herbicides which target the protox enzyme (alsoreferred to as “protox inhibitors”).

Other examples of herbicide-tolerance traits that could be combined withherbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., GAT and HRA plants) include thoseconferring tolerance to at least one herbicide in a plant such as, forexample, a maize plant or horseweed. Herbicide-tolerant weeds are knownin the art, as are plants that vary in their tolerance to particularherbicides. See, e.g., Green and Williams (2004) “Correlation of Corn(Zea mays) Inbred Response to Nicosulfuron and Mesotrione,” posterpresented at the WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12,2004; Green (1998) Weed Technology 12: 474-477; Green and Ulrich (1993)Weed Science 41: 508-516. The trait(s) responsible for these tolerancescan be combined by breeding or via other methods withherbicide-tolerance polynucleotides of the glyphosate/ALSinhibitor-tolerant plants (i.e., GAT and HRA plants) to provide a plantof the invention as well as methods of use thereof.

In this manner, the invention provides plants that are more tolerant toglyphosate and other ALS inhibitor chemistries and also provides plantsthat are more tolerant to the herbicide for which each of the traitsdiscussed above confers tolerance. Accordingly, the invention providesmethods for growing a crop (i.e., for selectively controlling weeds inan area of cultivation) that comprise treating an area of interest(e.g., a field or area of cultivation) with at least one herbicide towhich the plant of the invention is tolerant, such as for example,glyphosate, an ALS inhibitor chemistry, a mixture of ALS inhibitorchemistries, or a mixture of glyphosate and ALS inhibitor chemistry. Insome embodiments, methods of the invention further comprise treatmentwith additional herbicides to which the plant of the invention istolerant. In such embodiments, generally the methods of the inventionpermit selective control of weeds without significantly damaging thecrop. As used herein, an “area of cultivation” comprises any region inwhich one desires to grow a plant. Such areas of cultivations include,but are not limited to, a field in which a plant is cultivated (such asa crop field, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc), a greenhouse, a growth chamber,etc.

Herbicide-tolerant traits can also be combined with at least one othertrait to produce plants of the present invention that further comprise avariety of desired trait combinations including, but not limited to,traits desirable for animal feed such as high oil content (e.g., U.S.Pat. No. 6,232,529); balanced amino acid content (e.g., hordothionins(U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409; U.S.Pat. No. 5,850,016); barley high lysine (Williamson et al. (1987) Eur.J. Biochem. 165: 99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988)Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); thedisclosures of which are herein incorporated by reference. Desired traitcombinations also include LLNC (low linolenic acid content; see, e.g.,Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH(high oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J.Agric. Food Chem. 53: 5326-5330).

Herbicide-tolerant traits of interest can also be combined with otherdesirable traits such as, for example, fumonisim detoxification genes(U.S. Pat. No. 5,792,931), avirulence and disease resistance genes(Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable forprocessing or process products such as modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combineherbicide-tolerant polynucleotides with polynucleotides providingagronomic traits such as male sterility (e.g., see U.S. Pat. No.5.583,210), stalk strength, flowering time, or transformation technologytraits such as cell cycle regulation or gene targeting (e.g., WO99/61619, WO 00/17364, and WO 99/25821); the disclosures of which areherein incorporated by reference.

In another embodiment, the herbicide-tolerant traits of interest canalso be combined with the Rcg1 sequence or biologically active variantor fragment thereof. The Rcg1 sequence is an anthracnose stalk rotresistance gene in corn. See, for example, U.S. patent application Ser.No. 11/397,153, 11/397,275, and 11/397,247, each of which is hereinincorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Various changes in phenotype are of interest including modifying thefatty acid composition in a plant, altering the amino acid content of aplant, altering a plant's pathogen defense mechanism, and the like.These results can be achieved by providing expression of heterologousproducts or increased expression of endogenous products in plants.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors in the plant. These changes result in a change in phenotypeof the transformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like. Agronomically important traits suchas oil, starch, and protein content can be genetically altered inaddition to using traditional breeding methods. Modifications includeincreasing content of oleic acid, saturated and unsaturated oils,increasing levels of lysine and sulfur, providing essential amino acids,and also modification of starch.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen et al. (1986)J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both ofwhich are herein incorporated by reference); and rice (Musumura et al.(1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Otheragronomically important genes encode latex, Floury 2, growth factors,seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266: 789;Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell78: 1089); and the like.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxybutryrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitateexpression of polyhydroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

II. Polynucleotide Constructs

In specific embodiments, one or more of the herbicide-tolerantpolynucleotides employed in the methods and compositions can be providedin an expression cassette for expression in the plant or other organismof interest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a herbicide-tolerance polynucleotide. “Operablylinked” is intended to mean a functional linkage between two or moreelements. For example, an operable linkage between a polynucleotide ofinterest and a regulatory sequence (e.g., a promoter) is functional linkthat allows for expression of the polynucleotide of interest. Operablylinked elements may be contiguous or non-contiguous. When used to referto the joining of two protein coding regions, by “operably linked” isintended that the coding regions are in the same reading frame. Whenused to refer to the effect of an enhancer, “operably linked” indicatesthat the enhancer increases the expression of a particularpolynucleotide or polynucleotides of interest. Where the polynucleotideor polynucleotides of interest encode a polypeptide, the encodedpolypeptide is produced at a higher level.

The cassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes. Such an expressioncassette is provided with a plurality of restriction sites and/orrecombination sites for insertion of the herbicide-tolerancepolynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containother genes, including other selectable marker genes. Where a cassettecontains more than one polynucleotide, the polynucleotides in thecassette may be transcribed in the same direction or in differentdirections (also called “divergent” transcription).

An expression cassette comprising a herbicide-tolerance polynucleotidewill include in the 5′-3′ direction of transcription a transcriptionaland translational initiation region (i.e., a promoter), aherbicide-tolerance polynucleotide (e.g., a GAT polynucleotide, a ALSinhibitor-tolerant polynucleotide, an HRA polynucleotide, or anycombination thereof, etc.), and a transcriptional and translationaltermination region (i.e., termination region) functional in plants orthe other organism of interest. Accordingly, plants having suchexpression cassettes are also provided. The regulatory regions (i.e.,promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the herbicide-tolerance polynucleotide maybe native (i.e., analogous) to the host cell or to each other.Alternatively, the regulatory regions and/or the herbicide-tolerancepolynucleotide of the invention may be heterologous to the host cell orto each other. As used herein, “heterologous” in reference to a sequenceis a sequence that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same (i.e., analogous) species, one or bothare substantially modified from their original form and/or genomiclocus, or the promoter is not the native promoter for the operablylinked polynucleotide.

While it may be optimal to express polynucleotides using heterologouspromoters, native promoter sequences may be used. Such constructs canchange expression levels and/or expression patterns of the encodedpolypeptide in the plant or plant cell. Expression levels and/orexpression patterns of the encoded polypeptide may also be changed as aresult of an additional regulatory element that is part of theconstruct, such as, for example, an enhancer. Thus, the phenotype of theplant or cell can be altered even though a native promoter is used.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked herbicide-tolerancepolynucleotide of interest, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the herbicide-tolerance polynucleotide of interest, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions, or can be obtainedfrom plant genes such as the Solanum tuberosum proteinase inhibitor IIgene. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144;Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al.(1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.

A number of promoters can be used in the practice of the invention,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome.The polynucleotides of interest can be combined with constitutive,tissue-preferred, or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990)Plant Cell 2: 163-171); the maize actin promoter; the ubiquitin promoter(see, e.g., Christensen et al. (1989) Plant Mol. Biol. 12: 619-632;Christensen et al. (1992) Plant Mol. Biol. 18: 675-689; Callis et al.(1995) Genetics 139: 921-39); pEMU (Last et al. (1991) Theor. Appl.Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730);ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, those described in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611. Some promoters show improvedexpression when they are used in conjunction with a native 5′untranslated region and/or other elements such as, for example, anintron. For example, the maize ubiquitin promoter is often placedupstream of a polynucleotide of interest along with at least a portionof the 5′ untranslated region of the ubiquitin gene, including the firstintron of the maize ubiquitin gene.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter for which application of the chemicalinduces gene expression or the promoter may be a chemical-repressiblepromoter for which application of the chemical represses geneexpression. Chemical-inducible promoters are known in the art andinclude, but are not limited to, the maize In2-2 promoter, which isactivated by benzenesulfonamide herbicide safeners, the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides, and the tobacco PR-1a promoter,which is activated by salicylic acid. Other chemical-regulated promotersof interest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J.14(2): 247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), hereinincorporated by reference.

Tissue-preferred promoters can be utilized to target enhancedherbicide-tolerance polypeptide expression within a particular planttissue. Tissue-preferred promoters include Yamamoto et al. (1997) PlantJ. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3): 337-343; Russellet al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996)Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam(1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993)Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl.Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al. (1993) PlantJ. 4(3): 495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) PlantPhysiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993)Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc.Natl. Acad. Sci. USA 90(20): 9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3): 433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2): 343-350). The TR1′ gene, fused tonptII(neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4): 759-772); androlB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4): 681-691. Seealso U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10: 108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference.

Additional promoters of interest include the SCP1 promoter (U.S. Pat.No. 6,072,050), the HB2 promoter (U.S. Pat. No. 6,177,61 1) and the SAMSpromoter (US20030226166 and SEQ ID NO: 87 and biologically activevariants and fragments thereof); each of which is herein incorporated byreference. In addition, as discussed elsewhere herein, various enhancerscan be used with these promoters including, for example, the ubiquitinintron (i.e, the maize ubiquitin intron 1 (see, for example, NCBIsequence S94464), the omega enhancer or the omega prime enhancer (Gallieet al. (1989) Molecular Biology of RNA ed. Cech (Liss, N.Y.) 237-256 andGallie et al. Gene (1987) 60:217-25), or the 35S enhancer; each of whichis incorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42),and yellow fluorescent protein (PhiYFP from Evrogen, see, Bolte et al.(2004) J. Cell Science 117: 943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318;Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge etal. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen(1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993)Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell.Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidtet al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention, includingthe GAT gene and/or HRA gene.

Methods are known in the art of increasing the expression level of apolypeptide of the invention in a plant or plant cell, for example, byinserting into the polypeptide coding sequence one or two G/C-richcodons (such as GCG or GCT) immediately adjacent to and downstream ofthe initiating methionine ATG codon. Where appropriate, thepolynucleotides may be optimized for increased expression in thetransformed plant. That is, the polynucleotides can be synthesizedsubstituting in the polypeptide coding sequence one or more codons whichare less frequently utilized in plants for codons encoding the sameamino acid(s) which are more frequently utilized in plants, andintroducing the modified coding sequence into a plant or plant cell andexpressing the modified coding sequence. See, for example, Campbell andGowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferredcodon usage. Methods are available in the art for synthesizingplant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498,herein incorporated by reference. Embodiments comprising suchmodifications are also a feature of the invention.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. “Enhancers” such as the CaMV 35S enhancer may also beused (see, e.g., Benfey et al. (1990) EMBO J. 9: 1685-96), or otherenhancers may be used. For example, the sequence set forth in SEQ ID NO:1, 72, 79, 84, 85, 88, or 89 or a biologically active variant orfragment thereof can be used. See, also, U.S. Utility Application No.______, entitled “Methods and Compositions for the Expression of aPolynucleotide of Interest”, filed concurrently herewith, and hereinincorporated by reference in its entirety. The term “promoter” isintended to mean a regulatory region of DNA comprising a transcriptionalinitiation region, which in some embodiments, comprises a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. The promoter can further be operably linked to additionalregulatory elements that influence transcription, including, but notlimited to, introns, 5′ untranslated regions, and enhancer elements. Asused herein, an “enhancer sequence,” “enhancer domain,” “enhancerelement,” or “enhancer,” when operably linked to an appropriatepromoter, will modulate the level of transcription of an operably linkedpolynucleotide of interest. Biologically active fragments and variantsof the enhancer domain may retain the biological activity of modulating(increase or decrease) the level of transcription when operably linkedto an appropriate promoter.

Fragments of a polynucleotide for the enhancer domain or a promoter mayrange from at least about 50 nucleotides, about 100 nucleotides, about150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 350 nucleotides, about 400 nucleotides, about 450nucleotides, about 500 nucleotides, and up to the full-length nucleotidesequence of the invention for the enhancer domain of the invention. Inother embodiments, a fragment of the enhancer domain comprises a lengthof about 50 to about 100, 100 to about 150, 150 to about 200, 200 toabout 250, about 250 to about 300, about 300 to about 350, about 350 toabout 400, about 400 to about 450, about 450 to about 500, about 500 toabout 535 nucleotides. Generally, variants of a particularpolynucleotides of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particularpolynucleotides as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofan enhancer or a promoter may differ from that sequence by as few as1-15 nucleic acid residues, as few as 1 -10, such as 6-10, as few as 10,9, 8, 7, 6, 5, 4, 3, 2, or even 1 nucleic acid residue. Such activevariants and fragments will continue to modulate transcription.

Multiple copies of the enhancer domain or active variants and fragmentsthereof can be operably linked to a promoter. In specific embodiment,the chimeric transcriptional regulatory control region comprises atleast 1, 2, 3, 4, 5, 6, 7 or more copies of the enhancer domain. Infurther embodiments, the enhancer domain employed does not comprise thesequence set forth in SEQ ID NO:5. In addition, the enhancer can beorientated in either orientation (i.e., sense or reverse).

The distance between the promoter and the enhancer domain can vary, solong as the chimeric transcriptional regulatory region continues todirect transcription of the operably linked polynucleotide of interestin the desired manner. For example, an enhancer domain can be positionedat least about 10000 to about 15000, about 10000 to about a 9000, about9000 to about 8000, about 8000 to about 7000, about 7000 to about 6000,about 6000 to about 5000, about 5000 to about 4000, about 4000 to about3000, about 3000 to about 2000, about 2000 to about 1000, about 1000 toabout 500, about 500 to about 250, about 250 to immediately adjacent tothe promoter. It is further recognized that one or more copies of theenhancer can be placed upstream (5′) of the promoter or alternatively,one or more copies of the enhancer can be located 3′ to the promoter. Inspecific embodiments, when located 3′ of the promoter, the enhancer isdownstream of the terminator region. In still further embodiments, oneor more of the enhancers can be arranged either in the 5′ or 3′orientation (as shown in SEQ ID NO: 1 or 72) or in the 3′ to 5′orientation.

If multiple enhancers are employed, the enhancers can be positioned inthe construct with respect to the promoter such that the desired affecton expression is achieved. For example, the enhances can be immediatelyadjacent to each other or at least between 1 to 100, 100 to 300, 300 to500, 500 to 1000 nucleotides apart.

It is further recognized that the enhancer employed in the invention canbe positioned in a DNA construct between and operably linked to a firstand a second promoter. In such embodiments, the enhancer allows for amodulation in expression of both the first and the second promoters froma divergent direction. Exemplary, but non-limiting, examples of such DNAconstructs comprise in the 5′ to 3′ or 3′ to 5′ orientation: a firstpolynucleotide of interest operably linked to a first promoter, operablylinked to at least one copy of an enhancer of the invention, operablylinked to a second promoter, operably linked to a second polynucleotideof interest. In specific embodiments, the enhancer sequence isheterologous to the first and the second enhancer sequence. In otherembodiments, the first promoter is operably linked to a polynucleotideencoding an ALS inhibitor and the second promoter is operably linked toa polynucleotide encoding a polypeptide that confers tolerance toglyphosate. Such polynucleotides are disclosed elsewhere herein.

The expression cassette may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154: 9-20), and human immunoglobulin heavy-chainbinding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also,Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

In preparing the expression cassette, the various polynucleotidefragments may be manipulated, so as to provide for sequences to be inthe proper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join thefragments or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous material such asthe removal of restriction sites, or the like. For this purpose, invitro mutagenesis, primer repair, restriction, annealing,resubstitutions, e.g., transitions and transversions, may be involved.Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully, for example, inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor) (also known as“Maniatis”).

In some embodiments, the polynucleotide of interest is targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J.Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg Biomemb. 22(6): 789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36): 27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol.Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relieson particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride et al. (1994)Proc. Natl. Acad. Sci. USA 91: 7301-7305.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotide of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

“Gene” refers to a polynucleotide that expresses a specific protein,generally including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence(i.e., the portion of the sequence that encodes the specific protein).“Native gene” refers to a gene as found in nature, generally with itsown regulatory sequences. A “transgene” is a gene that has beenintroduced into the genome by a transformation procedure. Accordingly, a“transgenic plant” is a plant that contains a transgene, whether thetransgene was introduced into that particular plant by transformation orby breeding; thus, descendants of an originally-transformed plant areencompassed by the definition.

III. Methods of Introducing

The plants of the invention are generated by introducing a polypeptideor polynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, virus-mediatedmethods, and breeding.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell (i.e., monocot or dicot) targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83: 5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722),and ballistic particle acceleration (see, for example, U.S. Pat. Nos.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6: 923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674(soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (maize);Fromm et al. (1990) Biotechnology 8: 833-839 (maize); protocolspublished electronically by “IP.com” under the permanent publicationidentifiers IPCOM000033402D, IPCOM000033402D, and IPCOM000033402D andavailable at the “IP.com” website (cotton); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, herbicide-tolerance or other desirablesequences can be provided to a plant using a variety of transienttransformation methods. Such transient transformation methods include,but are not limited to, the introduction of the polypeptide or variantsand fragments thereof directly into the plant or the introduction of atranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci.44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107: 775-784, all ofwhich are herein incorporated by reference. Alternatively, aherbicide-tolerance polynucleotide can be transiently transformed intothe plant using techniques known in the art. Such techniques includeviral vector system and the precipitation of the polynucleotide in amanner that precludes subsequent release of the DNA. Thus, thetranscription from the particle-bound DNA can occur, but the frequencywith which it is released to become integrated into the genome isgreatly reduced. Such methods include the use particles coated withpolyethylimine (PEI; Sigma #P3143).

In other embodiments, polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a nucleotide construct within a viral DNAor RNA molecule. It is recognized that a polypeptide of interest may beinitially synthesized as part of a viral polyprotein, which later may beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Further, it is recognized that useful promoters mayinclude promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing apolypeptide encoded thereby, involving viral DNA or RNA molecules, areknown in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5: 209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,a polynucleotide can be contained in transfer cassette flanked by twonon-recombinogenic recombination sites. The transfer cassette isintroduced into a plant having stably incorporated into its genome atarget site which is flanked by two non-recombinogenic recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5: 81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

In specific embodiments, a polypeptide or the polynucleotide of interestis introduced into the plant cell. Subsequently, a plant cell having theintroduced sequence of the invention is selected using methods known tothose of skill in the art such as, but not limited to, Southern blotanalysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plantor plant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or activity of polypeptides in the plant. Plantforming conditions are well known in the art and discussed brieflyelsewhere herein.

It is also recognized that the level and/or activity of a polypeptide ofinterest may be modulated by employing a polynucleotide that is notcapable of directing, in a transformed plant, the expression of aprotein or an RNA. For example, the polynucleotides of the invention maybe used to design polynucleotide constructs that can be employed inmethods for altering or mutating a genomic nucleotide sequence in anorganism. Such polynucleotide constructs include, but are not limitedto, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96: 8774-8778; herein incorporated by reference.

It is therefore recognized that methods of the present invention do notdepend on the incorporation of the entire polynucleotide into thegenome, only that the plant or cell thereof is altered as a result ofthe introduction of the polynucleotide into a cell. In one embodiment ofthe invention, the genome may be altered following the introduction ofthe polynucleotide into a cell. For example, the polynucleotide, or anypart thereof, may incorporate into the genome of the plant. Alterationsto the genome of the present invention include, but are not limited to,additions, deletions, and substitutions of nucleotides into the genome.While the methods of the present invention do not depend on additions,deletions, and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions, or substitutions comprisesat least one nucleotide.

Plants of the invention may be produced by any suitable method,including breeding. Plant breeding can be used to introduce desiredcharacteristics (e.g., a stably incorporated transgene or a geneticvariant or genetic alteration of interest) into a particular plant lineof interest, and can be performed in any of several different ways.Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other elite inbred line having one ormore desirable characteristics (i.e., having stably incorporated apolynucleotide of interest, having a modulated activity and/or level ofthe polypeptide of interest, etc.) which complements the elite plantline of interest. If the two original parents do not provide all thedesired characteristics, other sources can be included in the breedingpopulation. In the pedigree method, superior plants are selfed andselected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection is practiced: F1→F2; F2→F3; F3→F4; F4→F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci. Various techniques known in the art can be used tofacilitate and accelerate the breeding (e.g., backcrossing) process,including, for example, the use of a greenhouse or growth chamber withaccelerated day/night cycles, the analysis of molecular markers toidentify desirable progeny, and the like.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. As discussed previously, backcrossing can be used totransfer one or more specifically desirable traits from one line, thedonor parent, to an inbred called the recurrent parent, which hasoverall good agronomic characteristics yet lacks that desirable trait ortraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, an F1, such as a commercial hybrid, is created. This commercialhybrid may be backcrossed to one of its parent lines to create a BC1 orBC2. Progeny are selfed and selected so that the newly developed inbredhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the non-recurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newhybrids and breeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of an inbred line of interest comprising the stepsof crossing a plant from the inbred line of interest with a donor plantcomprising at least one mutant gene or transgene conferring a desiredtrait (e.g., herbicide tolerance), selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1 progeny plant to a plant of the inbredline of interest. This method may further comprise the step of obtaininga molecular marker profile of the inbred line of interest and using themolecular marker profile to select for a progeny plant with the desiredtrait and the molecular marker profile of the inbred line of interest.In the same manner, this method may be used to produce an FI hybrid seedby adding a final step of crossing the desired trait conversion of theinbred line of interest with a different plant to make F1 hybrid seedcomprising a mutant gene or transgene conferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission ofuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” Fehr, 1993Macmillan Publishing Company the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

IV. Methods of Modulating Expression

In some embodiments, the activity and/or level of the polypeptide ismodulated (i.e., increased or decreased). An increase in the leveland/or activity of the polypeptide can be achieved by providing thepolypeptide to the plant. As discussed elsewhere herein, many methodsare known the art for providing a polypeptide to a plant including, butnot limited to, direct introduction of the polypeptide into the plant,introducing into the plant (transiently or stably) a polynucleotideconstruct encoding a polypeptide having the desired activity. It is alsorecognized that the methods of the invention may employ a polynucleotidethat is not capable of directing, in the transformed plant, theexpression of a protein or an RNA. Thus, the level and/or activity of apolypeptide may be modulated by altering the gene encoding thepolypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350;Zarling et al., PCT/US93/03868. Therefore mutagenized plants that carrymutations in genes, where the mutations increase expression of the geneor increase the activity of the encoded polypeptide are provided.

In other embodiments, the activity and/or level of a polypeptide isreduced or eliminated by introducing into a plant a polynucleotide thatinhibits the level or activity of the polypeptide. The polynucleotidemay inhibit the expression of the polypeptide directly, by preventingtranslation of the corresponding messenger RNA, or indirectly, byencoding a polypeptide that inhibits the transcription or translation ofa gene encoding the protein. Methods for inhibiting or eliminating theexpression of a gene in a plant are well known in the art, and any suchmethod may be used in the present invention to inhibit the expression ofa gene in a plant. In other embodiments of the invention, the activityof a polypeptide is reduced or eliminated by transforming a plant cellwith a sequence encoding a polypeptide that inhibits the activity of thepolypeptide. In other embodiments, the activity of a polypeptide may bereduced or eliminated by disrupting the gene encoding the polypeptide.The invention encompasses mutagenized plants that carry mutations ingenes of interest, where the mutations reduce expression of the gene orinhibit the activity of the encoded polypeptide.

Reduction of the activity of specific genes (also known as genesilencing or gene suppression) is desirable for several aspects ofgenetic engineering in plants. Many techniques for gene silencing arewell known to one of skill in the art, including, but not limited to,antisense technology (see, e.g., Sheehy et al. (1988) Proc. Natl. Acad.Sci. USA 85: 8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and5,759,829); cosuppression (e.g., Taylor (1997) Plant Cell 9: 1245;Jorgensen (1990) Trends Biotech. 8(12): 340-344; Flavell (1994) Proc.Natl. Acad. Sci. USA 91: 3490-3496; Finnegan et al. (1994)Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen. Genet.244: 230-241); RNA interference (Napoli et al. (1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13: 139-141;Zamore et al. (2000) Cell 101: 25-33; and Montgomery et al. (1998) Proc.Natl. Acad. Sci. USA 95: 15502-15507), virus-induced gene silencing(Burton et al. (2000) Plant Cell 12: 691-705; and Baulcombe (1999) Curr.Op. Plant Bio. 2: 109-113); target-RNA-specific ribozymes (Haseloffetal. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000)Nature 407: 319-320; WO 99/53050; WO 02/00904; WO 98/53083; Chuang andMeyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijket al. (2002) Plant Physiol. 129: 1723-1731; Waterhouse and Helliwell(2003) Nat. Rev. Genet. 4: 29-38; Pandolfini et al. BMC Biotechnology 3:7, U.S. Patent Publication No. 20030175965; Panstruga et al. (2003) Mol.Biol. Rep. 30: 135-140; Wesley et al. (2001) Plant J. 27: 581-590; Wangand Waterhouse (2001) Curr. Opin. Plant Biol. 5: 146-150; U.S. PatentPublication No. 20030180945; and WO 02/00904, all of which are hereinincorporated by reference); ribozymes (Steinecke et al. (1992) EMBO J.11: 1525; and Perriman et al. (1993) Antisense Res. Dev. 3: 253);oligonucleotide-mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); transposon tagging (Maes et al. (1999)Trends Plant Sci. 4: 90-96; Dharmapuri and Sonti (1999) FEMS Microbiol.Lett. 179: 53-59; Meissner et al. (2000) Plant J. 22: 265-274; Phogat etal. (2000) J. Biosci. 25: 57-63; Walbot (2000) Curr. Opin. Plant Biol.2: 103-107; Gai et al. (2000) Nucleic Acids Res. 28: 94-96; Fitzmauriceet al. (1999) Genetics 153: 1919-1928; Bensen et al. (1995) Plant Cell7: 75-84; Mena et al. (1996) Science 274: 1537-1540; and U.S. Pat. No.5,962,764); each of which is herein incorporated by reference; and othermethods or combinations of the above methods known to those of skill inthe art.

It is recognized that antisense constructions complementary to at leasta portion of the messenger RNA (mRNA) for a polynucleotide of interestcan be constructed. Antisense nucleotides are constructed to hybridizewith the corresponding mRNA. Modifications of the antisense sequencesmay be made as long as the sequences hybridize to and interfere withexpression of the corresponding mRNA. In this manner, antisenseconstructions having at least 70%, optimally 80%, more optimally 85%sequence identity to the corresponding antisensed sequences may be used.Furthermore, portions of the antisense nucleotides may be used todisrupt the expression of the target gene. Generally, sequences of atleast 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450,500, 550, or greater may be used.

Polynucleotides may also be used in the sense orientation to suppressthe expression of endogenous genes in plants. Methods for suppressinggene expression in plants using polynucleotides in the sense orientationare known in the art. The methods generally involve transforming plantswith a DNA construct comprising a promoter that drives expression in aplant operably linked to at least a portion of a polynucleotide thatcorresponds to the transcript of the endogenous gene. Typically, such anucleotide sequence has substantial sequence identity to the sequence ofthe transcript of the endogenous gene, generally greater than about 65%,85%, or 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323; herein incorporated by reference. Thus, many methods may beused to reduce or eliminate the activity of a polypeptide. More than onemethod may be used to reduce the activity of a single polypeptide. Inaddition, combinations of methods may be employed to reduce or eliminatethe activity of a polypeptide.

In one embodiment, the expression level of a polypeptide may be measureddirectly, for example, by assaying for the level of the polynucleotideor polypeptide or a known metabolite in the plant (e.g., by assaying forthe level of N-acetylglyphosate (“NAG”) in a plant containing a GATgene), or indirectly, for example, by evaluating the plant containing itfor the trait to be conferred by the polypeptide, e.g., herbicideresistance.

V. Methods of Controlling Weeds

Methods are provided for controlling weeds in an area of cultivation,preventing the development or the appearance of herbicide resistantweeds in an area of cultivation, producing a crop, and increasing cropsafety. The term “controlling,” and derivations thereof, for example, asin “controlling weeds” refers to one or more of inhibiting the growth,germination, reproduction, and/or proliferation of; and/or killing,removing, destroying, or otherwise diminishing the occurrence and/oractivity of a weed.

The glyphosate/ALS inhibitor plants of the invention display a modifiedtolerance to herbicides and therefore allow for the application of oneor more herbicides at rates that would significantly damage controlplants and further allow for the application of combinations ofherbicides at lower concentrations than normally applied which stillcontinue to selectively control weeds. In addition, the glyphosate/ALSinhibitor-tolerant plants of the invention can be used in combinationwith herbicide blends technology and thereby make the application ofchemical pesticides more convenient, economical, and effective for theproducer.

The methods of the invention comprise planting the area of cultivationwith glyphosate/ALS inhibitor-tolerant crop seeds or plants of theinvention, and in specific embodiments, applying to any crop, crop part,weed or area of cultivation thereof an effective amount of a herbicideof interest. It is recognized that the herbicide can be applied beforeor after the crop is planted in the area of cultivation. Such herbicideapplications can include an application of glyphosate, an ALS inhibitorchemistry, or any combination thereof. In specific embodiments, amixture of ALS inhibitor chemistry in combination with glyphosate isapplied to the glyphosate/ALS inhibitor-tolerant plant, wherein theeffective concentration of at least two of the ALS inhibitor chemistrieswould significantly damage an appropriate control plant. In onenon-limiting embodiment, the herbicide comprises at least one of asulfonylaminocarbonyltriazolinone; a triazolopyrimidine; apyrimidinyl(thio)benzoate; an imidazolinone; a triazine; and/or aphosphinic acid.

In another non-limiting embodiment, the combination of herbicidescomprises glyphosate, imazapyr, chlorimuron-ethyl, quizalofop, andfomesafen, wherein said effective amount is tolerated by the crop andcontrols weeds. As disclosed elsewhere herein, any effective amount ofthese herbicides can be applied. In specific embodiments, thiscombination of herbicides comprises an effective amount of glyphosatecomprising about 1110 to about 1130 g ai/hectare; an effective amount ofimazapyr comprising about 7.5 to about 27.5 g ai/hectare; an effectiveamount of chlorimuron-ethyl comprising about 7.5 to about 27.5 gai/hectare; an effective amount of quizalofop comprising about 50 toabout 70 g ai/hectare; and, an effective amount of fomesafen comprisingabout 240 to about 260 g ai/hectare.

In other embodiments, at least a combination of two herbicides areapplied, wherein the combination does not include glyphosate. In otherembodiments, at least one ALS inhibitor and glyphosate is applied to theplant. More details regarding the various herbicide combinations thatcan be employed in the methods of the invention are discussed elsewhereherein.

In one embodiment, the method of controlling weeds comprises plantingthe area with a glyphosate/ALS inhibitor-tolerant crop seeds or plantand applying to the crop, crop part, seed of said crop or the area undercultivation, an effective amount of a herbicide, wherein said effectiveamount comprises

i) an amount that is not tolerated by a first control crop when appliedto the first control crop, crop part, seed or the area of cultivation,wherein said first control crop expresses a first polynucleotide thatconfers tolerance to glyphosate and does not express a secondpolynucleotide that encodes an ALS inhibitor-tolerant polypeptide;

ii) an amount that is not tolerated by a second control crop whenapplied to the second crop, crop part, seed or the area of cultivation,wherein said second control crop expresses the second polynucleotide anddoes not express the first polynucleotide; and,

iii) an amount that is tolerated when applied to the glyphosate/ALSinhibitor-tolerant crop, crop part, seed, or the area of cultivationthereof. The herbicide can comprise a combination of herbicides thateither includes or does not include glyphosate. In specific embodiments,the combination of herbicides comprises ALS inhibitor chemistries asdiscussed in further detail below.

In another embodiment, the method of controlling weeds comprisesplanting the area with a glyphosate/ALS inhibitor-tolerant crop seeds orplant and applying to the crop, crop part, seed of said crop or the areaunder cultivation, an effective amount of a herbicide, wherein saideffective amount comprises a level that is above the recommended labeluse rate for the crop, wherein said effective amount is tolerated whenapplied to the glyphosate/ALS inhibitor-tolerant crop, crop part, seed,or the area of cultivation thereof. The herbicide applied can comprise acombination of herbicides that either includes or does not includeglyphosate. In specific embodiments, the combination of herbicidescomprises at least one ALS inhibitor chemistries as discussed in furtherdetail below. Further herbicides and combinations thereof that can beemployed in the various methods of the invention are discussed infurther detail below.

In another non-limiting embodiment, the herbicide applied in any methoddisclosed herein does not comprise glyphosate, chlorimuron-methyl,rimsulfuron, tribenuron-methyl or thifensufuron-methyl.

a. Types of Herbicides

Any herbicide can be applied to the glyphosate/ALS inhibitor-tolerantcrop, crop part, or the area of cultivation containing said crop plant.Classifications of herbicides (i.e., the grouping of herbicides intoclasses and subclasses) is well-known in the art and includesclassifications by HRAC (Herbicide Resistance Action Committee) and WSSA(the Weed Science Society of America) (see also, Retzinger andMallory-Smith (1997) Weed Technology 11: 384-393). An abbreviatedversion of the HRAC classification (with notes regarding thecorresponding WSSA group) is set forth below in Table 1.

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application), or by how they are taken up byor affect the plant. For example, thifensulfuron-methyl andtribenuron-methyl are applied to the foliage of a crop (e.g., maize) andare generally metabolized there, while rimsulfuron and chlorimuron-ethylare generally taken up through both the roots and foliage of a plant.“Mode of action” generally refers to the metabolic or physiologicalprocess within the plant that the herbicide inhibits or otherwiseimpairs, whereas “site of action” generally refers to the physicallocation or biochemical site within the plant where the herbicide actsor directly interacts. Herbicides can be classified in various ways,including by mode of action and/or site of action (see, e.g., Table 1).

Often, a herbicide-tolerance gene that confers tolerance to a particularherbicide or other chemical on a plant expressing it will also confertolerance to other herbicides or chemicals in the same class orsubclass, for example, a class or subclass set forth in Table 1. Thus,in some embodiments of the invention, a transgenic plant of theinvention is tolerant to more than one herbicide or chemical in the sameclass or subclass, such as, for example, an inhibitor of PPO, asulfonylurea, or a synthetic auxin.

Typically, the plants of the present invention can tolerate treatmentwith different types of herbicides (i.e., herbicides having differentmodes of action and/or different sites of action) as well as with higheramounts of herbicides than previously known plants, thereby permittingimproved weed management strategies that are recommended in order toreduce the incidence and prevalence of herbicide-tolerant weeds.Specific herbicide combinations can be employed to effectively controlweeds.

The invention thereby provides a transgenic crop plant which can beselected for use in crop production based on the prevalence ofherbicide-tolerant weed species in the area where the transgenic crop isto be grown. Methods are known in the art for assessing the herbicidetolerance of various weed species. Weed management techniques are alsoknown in the art, such as for example, crop rotation using a crop thatis tolerant to a herbicide to which the local weed species are nottolerant. A number of entities monitor and publicly report the incidenceand characteristics of herbicide-tolerant weeds, including the HerbicideResistance Action Committee (HRAC), the Weed Science Society of America,and various state agencies (see, e.g., see, for example, herbicidetolerance scores for various broadleaf weeds from the 2004 IllinoisAgricultural Pest Management Handbook), and one of skill in the artwould be able to use this information to determine which crop andherbicide combinations should be used in a particular location.

These entities also publish advice and guidelines for preventing thedevelopment and/or appearance of and controlling the spread of herbicidetolerant weeds (see, e.g., Owen and Hartzler (2004), 2005 HerbicideManual for Agricultural Professionals, Pub. WC 92 Revised (Iowa StateUniversity Extension, Iowa State University of Science and Technology,Ames, Iowa); Weed Control for Corn, Soybeans, and Sorghum, Chapter 2 of“2004 Illinois Agricultural Pest Management Handbook” (University ofIllinois Extension, University of Illinois at Urbana-Champaign, Ill.));Weed Control Guide for Field Crops, MSU Extension Bulletin E434(Michigan State University, East Lansing, Mich.)). TABLE 1 Abbreviatedversion of HRAC Herbicide Classification I. ALS Inhibitors (WSSA Group2) A. Sulfonylureas 1. Azimsulfuron 2. Chlorimuron-ethyl 3.Metsulfuron-methyl 4. Nicosulfuron 5. Rimsulfuron 6. Sulfometuron-methyl7. Thifensulfuron-methyl 8. Tribenuron-methyl 9. Amidosulfuron 10.Bensulfuron-methyl 11. Chlorsulfuron 12. Cinosulfuron 13.Cyclosulfamuron 14. Ethametsulfuron-methyl 15. Ethoxysulfuron 16.Flazasulfuron 17. Flupyrsulfuron-methyl 18. Foramsulfuron 19.Imazosulfuron 20. Iodosulfuron-methyl 21. Mesosulfuron-methyl 22.Oxasulfuron 23. Primisulfuron-methyl 24. Prosulfuron 25.Pyrazosulfuron-ethyl 26. Sulfosulfuron 27. Triasulfuron 28.Trifloxysulfuron 29. Triflusulfuron-methyl 30. Tritosulfuron 31.Halosulfuron-methyl 32. Flucetosulfuron B.Sulfonylaminocarbonyltriazolinones 1. Flucarbazone 2. Procarbazone C.Triazolopyrimidines 1. Cloransulam-methyl 2. Flumetsulam 3. Diclosulam4. Florasulam 5. Metosulam 6. Penoxsulam 7. Pyroxsulam D.Pyrimidinyloxy(thio)benzoates 1. Bispyribac 2. Pyriftalid 3.Pyribenzoxim 4. Pyrithiobac 5. Pyriminobac-methyl E. Imidazolinones 1.Imazapyr 2. Imazethapyr 3. Imazaquin 4. Imazapic 5.Imazamethabenz-methyl 6. Imazamox II. Other Herbicides - ActiveIngredients/ Additional Modes of Action A. Inhibitors of Acetyl CoAcarboxylase (ACCase) (WSSA Group 1) 1. Aryloxyphenoxypropionates(‘FOPs’) a. Quizalofop-P-ethyl b. Diclofop-methyl c.Clodinafop-propargyl d. Fenoxaprop-P-ethyl e. Fluazifop-P-butyl f.Propaquizafop g. Haloxyfop-P-methyl h. Cyhalofop-butyl i.Quizalofop-P-ethyl 2. Cyclohexanediones (‘DIMs’) a. Alloxydim b.Butroxydim c. Clethodim d. Cycloxydim e. Sethoxydim f. Tepraloxydim g.Tralkoxydim B. Inhibitors of Photosystem II - HRAC Group C1/WSSA Group5 1. Triazines a. Ametryne b. Atrazine c. Cyanazine d. Desmetryne e.Dimethametryne f. Prometon g. Prometryne h. Propazine i. Simazine j.Simetryne k. Terbumeton l. Terbuthylazine m. Terbutryne n. Trietazine 2.Triazinones a. Hexazinone b. Metribuzin c. Metamitron 3. Triazolinone a.Amicarbazone 4. Uracils a. Bromacil b. Lenacil c. Terbacil 5.Pyridazinones a. Pyrazon 6. Phenyl carbamates a. Desmedipham b.Phenmedipham C. Inhibitors of Photosystem II - HRAC Group C2/WSSA Group7 1. Ureas a. Fluometuron b. Linuron c. Chlorobromuron d. Chlorotolurone. Chloroxuron f. Dimefuron g. Diuron h. Ethidimuron i. Fenuron j.Isoproturon k. Isouron l. Methabenzthiazuron m. Metobromuron n.Metoxuron o. Monolinuron p. Neburon q. Siduron r. Tebuthiuron 2. Amidesa. Propanil b. Pentanochlor D. Inhibitors of Photosystem II - HRAC GroupC3/WSSA Group 6 1. Nitriles a. Bromofenoxim b. Bromoxynil C. Ioxynil 2.Benzothiadiazinone (Bentazon) a. Bentazon 3. Phenylpyridazines a.Pyridate b. Pyridafol E. Photosystem-I-electron diversion(Bipyridyliums) (WSSA Group 22) 1. Diquat 2. Paraquat F. Inhibitors ofPPO (protoporphyrinogen oxidase) (WSSA Group 14) 1. Diphenylethers a.Acifluorfen-Na b. Bifenox c. Chlomethoxyfen d. Fluoroglycofen-ethyl e.Fomesafen f. Halosafen g. Lactofen h. Oxyfluorfen 2. Phenylpyrazoles a.Fluazolate b. Pyraflufen-ethyl 3. N-phenylphthalimides a. Cinidon-ethylb. Flumioxazin c. Flumiclorac-pentyl 4. Thiadiazoles a.Fluthiacet-methyl b. Thidiazimin 5. Oxadiazoles a. Oxadiazon b.Oxadiargyl 6. Triazolinones a. Carfentrazone-ethyl b. Sulfentrazone 7.Oxazolidinediones a. Pentoxazone 8. Pyrimidindiones a. Benzfendizone b.Butafenicil 9. Others a. Pyrazogyl b. Profluazol G. Bleaching:Inhibition of carotenoid biosynthesis at the phytoene desaturase step(PDS) (WSSA Group 12) 1. Pyridazinones a. Norflurazon 2.Pyridinecarboxamides a. Diflufenican b. Picolinafen 3. Others a.Beflubutamid b. Fluridone c. Flurochloridone d. Flurtamone H. Bleaching:Inhibition of 4- hydroxyphenyl-pyruvate-dioxygenase (4-HPPD) (WSSA Group28) 1. Triketones a. Mesotrione b. Sulcotrione 2. Isoxazoles a.Isoxachlortole b. Isoxaflutole 3. Pyrazoles a. Benzofenap b. Pyrazoxyfenc. Pyrazolynate 4. Others a. Benzobicyclon I. Bleaching: Inhibition ofcarotenoid biosynthesis (unknown target) (WSSA Group 11 and 13) 1.Triazoles (WSSA Group 11) a. Amitrole 2. Isoxazolidinones (WSSA Group13) a. Clomazone 3. Ureas a. Fluometuron 3. Diphenylether a. AclonifenJ. Inhibition of EPSP Synthase 1. Glycines (WSSA Group 9) a. Glyphosateb. Sulfosate K. Inhibition of glutamine synthetase 1. Phosphinic Acidsa. Glufosinate-ammonium b. Bialaphos L. Inhibition of DHP(dihydropteroate) synthase (WSSA Group 18) 1. Carbamates a. Asulam M.Microtubule Assembly Inhibition (WSSA Group 3) 1. Dinitroanilines a.Benfluralin b. Butralin c. Dinitramine d. Ethalfluralin e. Oryzalin f.Pendimethalin g. Trifluralin 2. Phosphoroamidates a. Amiprophos-methylb. Butamiphos 3. Pyridines a. Dithiopyr b. Thiazopyr 4. Benzamides a.Pronamide b. Tebutam 5. Benzenedicarboxylic acids a. Chlorthal-dimethylN. Inhibition of mitosis/microtubule organization WSSA Group 23) 1.Carbamates a. Chlorpropham b. Propham c. Carbetamide O. Inhibition ofcell division (Inhibition of very long chain fatty acids as proposedmechanism; WSSA Group 15) 1. Chloroacetamides a. Acetochlor b. Alachlorc. Butachlor d. Dimethachlor e. Dimethanamid f. Metazachlor g.Metolachlor h. Pethoxamid i. Pretilachlor j. Propachlor k. Propisochlorl. Thenylchlor 2. Acetamides a. Diphenamid b. Napropamide c.Naproanilide 3. Oxyacetamides a. Flufenacet b. Mefenacet 4.Tetrazolinones a. Fentrazamide 5. Others a. Anilofos b. Cafenstrole c.Indanofan d. Piperophos P. Inhibition of cell wall (cellulose)synthesis 1. Nitriles (WSSA Group 20) a. Dichlobenil b. Chlorthiamid 2.Benzamides (isoxaben (WSSA Group 21)) a. Isoxaben 3.Triazolocarboxamides (flupoxam) a. Flupoxam Q. Uncoupling (membranedisruption): (WSSA Group 24) 1. Dinitrophenols a. DNOC b. Dinoseb c.Dinoterb R. Inhibition of Lipid Synthesis by other than ACCinhibition 1. Thiocarbamates (WSSA Group 8) a. Butylate b. Cycloate c.Dimepiperate d. EPTC e. Esprocarb f. Molinate g. Orbencarb h. Pebulatei. Prosulfocarb j. Benthiocarb k. Tiocarbazil l. Triallate m. Vernolate2. Phosphorodithioates a. Bensulide 3. Benzofurans a. Benfuresate b.Ethofumesate 4. Halogenated alkanoic acids (WSSA Group 26) a. TCA b.Dalapon c. Flupropanate S. Synthetic auxins (IAA-like) (WSSA Group 4) 1.Phenoxycarboxylic acids a. Clomeprop b. 2,4-D c. Mecoprop 2. Benzoicacids a. Dicamba b. Chloramben c. TBA 3. Pyridine carboxylic acids a.Clopyralid b. Fluroxypyr c. Picloram d. Tricyclopyr 4. Quinolinecarboxylic acids a. Quinclorac b. Quinmerac 5. Others (benazolin-ethyl)a. Benazolin-ethyl T. Inhibition of Auxin Transport 1. Phthalamates;semicarbazones (WSSA Group 19) a. Naptalam b. Diflufenzopyr-Na U. OtherMechanism of Action 1. Arylaminopropionic acids a.Flamprop-M-methyl/-isopropyl 2. Pyrazolium a. Difenzoquat 3.Organoarsenicals a. DSMA b. MSMA 4. Others a. Bromobutide b. Cinmethylinc. Cumyluron d. Dazomet e. Daimuron-methyl f. Dimuron g. Etobenzanid h.Fosamine i. Metam j. Oxaziclomefone k. Oleic acid l. Pelargonic acid m.Pyributicarb

In one embodiment, one ALS inhibitor or at least two ALS inhibitors areapplied to the glyphosate/ALS inhibitor-tolerant crop or area ofcultivation. In one non-limiting embodiment, the combination of ALSherbicides does not include glyphosate. The ALS inhibitor can be appliedat any effective rate that selectively controls weeds and does notsignificantly damage the crop. In specific embodiments, at least one ALSinhibitor is applied at a level that would significantly damage anappropriate control plant. In other embodiments, at least one ALSinhibitor is applied above the recommended label use rate for the crop.In still other embodiments, a mixture of ALS inhibitors is applied at alower rate than the recommended use rate and weeds continue to beselectively controlled. Herbicides that inhibit acetolactate synthase(also known as acetohydroxy acid synthase) and are therefore useful inthe methods of the invention include sulfonylureas as listed in Table 1,including agriculturally suitable salts (e.g., sodium salts) thereof;sulfonylaminocarbonyltriazolinones as listed in Table 1, includingagriculturally suitable salts (e.g., sodium salts) thereof;triazolopyrimidines as listed in Table 1, including agriculturallysuitable salts (e.g., sodium salts) thereof;pyrimidinyloxy(thio)benzoates as listed in Table 1, includingagriculturally suitable salts (e.g., sodium salts) thereof; andimidazolinones as listed in Table 1, including agriculturally suitablesalts (e.g., sodium salts) there. In some embodiments, methods of theinvention comprise the use of a sulfonylurea which is notchlorimuron-ethyl, chlorsulfuron, rimsulfuron, thifensulfuron-methyl, ortribenuron-methyl.

In still further methods, glyphosate, in combination with anotherherbicide of interest, can be applied to the glyphosate/ALSinhibitor-tolerant plants or their area of cultivation. Non-limitingexamples of glyphosate formations are set forth in Table 2. In specificembodiments, the glyphosate is in the form of a salt, such as, ammonium,isopropylammonium, potassium, sodium (including sesquisodium) ortrimesium (alternatively named sulfosate). In still further embodiments,a mixture of a synergistically effective amount of a combination ofglyphosate and an ALS inhibitor (such as a sulfonylurea) is applied tothe glyphosate/ALS inhibitor-tolerant plants or their area ofcultivation. TABLE 2 Glyphosate formulations comparisons. Active AcidAcid ingredient equivalent Apply: equivalent Herbicide by Registered perper fl oz/ per Trademark Manufacturer Salt gallon gallon acre acreRoundup Original Monsanto Isopropylamine 4 3 32 0.750 Roundup OriginalII Monsanto Isopropylamine 4 3 32 0.750 Roundup Original MAX MonsantoPotassium 5.5 4.5 22 0.773 Roundup UltraMax Monsanto Isopropylamine 53.68 26 0.748 Roundup UltraMax II Monsanto Potassium 5.5 4.5 22 0.773Roundup Weathermax Monsanto Potassium 5.5 4.5 22 0.773 TouchdownSyngenta Diammomium 3.7 3 32 0.750 Touchdown HiTech Syngenta Potassium6.16 5 20 0.781 Touchdown Total Syngenta Potassium 5.14 4.17 24 0.782Durango Dow AgroSciences Isopropylamine 5.4 4 24 0.750 Glyphomax DowAgroSciences Isopropylamine 4 3 32 0.750 Glyphomax Plus Dow AgroSciencesIsopropylamine 4 3 32 0.750 Glyphomax XRT Dow AgroSciencesIsopropylamine 4 3 32 0.750 Gly Star Plus Albaugh/Agri StarIsopropylamine 4 3 32 0.750 Gly Star 5 Albaugh/Agri Star Isopropylamine5.4 4 24 0.750 Gly Star Original Albaugh/Agri Star Isopropylamine 4 3 320.750 Gly-Flo Micro Flo Isopropylamine 4 3 32 0.750 Credit NufarmIsopropylamine 4 3 32 0.750 Credit Extra Nufarm Isopropylamine 4 3 320.750 Credit Duo Nufarm Isopro + monoamm. 4 3 32 0.750 Credit Duo ExtraNufarm Isopro + monoamm. 4 3 32 0.750 Extra Credit 5 NufarmIsopropylamine 5 3.68 26 0.748 Cornerstone Agriliance Isopropylamine 4 332 0.750 Cornerstone Plus Agriliance Isopropylamine 4 3 32 0.750 GlyfosCheminova Isopropylamine 4 3 32 0.750 Glyfos X-TRA CheminovaIsopropylamine 4 3 32 0.750 Rattler Helena Isopropylamine 4 3 32 0.750Rattler Plus Helena Isopropylamine 4 3 32 0.750 Mirage UAPIsopropylamine 4 3 32 0.750 Mirage Plus UAP Isopropylamine 4 3 32 0.750Glyphosate 41% Helm Agro USA Isopropylamine 4 3 32 0.750 BuccaneerTenkoz Isopropylamine 4 3 32 0.750 Buccaneer Plus Tenkoz Isopropylamine4 3 32 0.750 Honcho Monsanto Isopropylamine 4 3 32 0.750 Honcho PlusMonsanto Isopropylamine 4 3 32 0.750 Gly-4 Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 Gly-4 Plus Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 ClearOut 41 Chemical Products Isopropylamine4 3 32 0.750 Tech. ClearOut 41 Plus Chemical Products Isopropylamine 4 332 0.750 Tech. Spitfire Control Soultions Isopropylamine 4 3 32 0.750Spitfire Plus Control Soultions Isopropylamine 4 3 32 0.750 Glyphosate 4FarmerSaver.com Isopropylamine 4 3 32 0.750 FS Glyphosate Plus GrowmarkIsopropylamine 4 3 32 0.750 Glyphosate Original Griffin, LLC.Isopropylamine 4 3 32 0.750

Thus, in some embodiments, a transgenic plant of the invention is usedin a method of growing a glyphosate/ALS inhibitor-tolerant crop by theapplication of herbicides to which the plant is tolerant. In thismanner, treatment with a combination of one of more herbicides whichinclude, but are not limited to: acetochlor, acifluorfen and its sodiumsalt, aclonifen, acrolein (2-propenal), alachlor, alloxydim, ametryn,amicarbazone, amidosulfuron, aminopyralid, amitrole, ammonium sulfamate,anilofos, asulam, atrazine, azimsulfuron, beflubutamid, benazolin,benazolin-ethyl, bencarbazone, benfluralin, benfuresate,bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap,bifenox, bilanafos, bispyribac and its sodium salt, bromacil,bromobutide, bromofenoxim, bromoxynil, bromoxynil octanoate, butachlor,butafenacil, butamifos, butralin, butroxydim, butylate, cafenstrole,carbetamide, carfentrazone-ethyl, catechin, chlomethoxyfen, chloramben,chlorbromuron, chlorflurenol-methyl, chloridazon, chlorimuron-ethyl,chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl,chlorthiamid, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim,clodinafop-propargyl, clomazone, clomeprop, clopyralid,clopyralid-olamine, cloransulam-methyl, CUH-35 (2-methoxyethyl2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl](3-fluoro-benzoyl)amino]carbonyl]-1-cyclohexene-1-carboxylate),cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim,cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropylesters and its dimethylammonium, diolamine and trolamine salts,daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and itsdimethylammonium, potassium and sodium salts, desmedipham, desmetryn,dicamba and its diglycolammonium, dimethylammonium, potassium and sodiumsalts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam,difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimethylarsinic acid and its sodium salt, dinitramine,dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC,endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl,flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl,fluridone, flurochloridone, fluroxypyr, flurtamone, fluthiacet-methyl,fomesafen, foramsulfuron, fosamine-ammonium, glufosinate,glufosinate-ammonium, glyphosate and its salts such as ammonium,isopropylammonium, potassium, sodium (including sesquisodium) andtrimesium (alternatively named sulfosate), halosulfuron-methyl,haloxyfop-etotyl, haloxyfop-methyl, hexazinone, HOK-201(N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)methyl]-4H-1,2,4-triazole-4-carboxamide),imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin,imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulfuron,indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate,ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole,isoxachlortole, lactofen, lenacil, linuron, maleic hydrazide, MCPA andits salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium,esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g.,MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters(e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide,mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron,metazachlor, methabenzthiazuron, methylarsonic acid and its calcium,monoammonium, monosodium and disodium salts, methyldymron, metobenzuron,metobromuron, metolachlor, S-metholachlor, metosulam, metoxuron,metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide,napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid,pendimethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone,pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen,pinoxaden, piperofos, pretilachlor, primisulfuron-methyl, prodiamine,profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop,propazine, propham, propisochlor, propoxycarbazone, propyzamide,prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole,pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl,pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac,quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine,simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl,sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron,tefuryltrione, tembotrione, tepraloxydim, terbacil, terburneton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl,triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane,trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl,tritosulfuron and vemolate.

Other suitable herbicides and agricultural chemicals are known in theart, such as, for example, those described in WO 2005/041654. Otherherbicides also include bioherbicides such as Alternaria destruensSimmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc., Drechsieramonoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz)Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Pucciniathlaspeos Schub. Combinations of various herbicides can result in agreater-than-additive (i.e., synergistic) effect on weeds and/or aless-than-additive effect (i.e. safening) on crops or other desirableplants. In certain instances, combinations of glyphosate with otherherbicides having a similar spectrum of control but a different mode ofaction will be particularly advantageous for preventing the developmentof resistant weeds. Herbicidally effective amounts of any particularherbicide can be easily determined by one skilled in the art throughsimple experimentation.

Herbicides may be classified into groups and/or subgroups as describedherein above with reference to their mode of action, or they may beclassified into groups and/or subgroups in accordance with theirchemical structure.

Sulfonamide herbicides have as an essential molecular structure featurea sulfonamide moiety (—S(O)₂NH—). As referred to herein, sulfonamideherbicides particularly comprise sulfonylurea herbicides,sulfonylaminocarbonyltriazolinone herbicides and triazolopyrimidineherbicides. In sulfonylurea herbicides the sulfonamide moiety is acomponent in a sulfonylurea bridge (—S(O)₂NHC(O)NH(R)—). In sulfonylureaherbicides the sulfonyl end of the sulfonylurea bridge is connectedeither directly or by way of an oxygen atom or an optionally substitutedamino or methylene group to a typically substituted cyclic or acyclicgroup. At the opposite end of the sulfonylurea bridge, the amino group,which may have a substituent such as methyl (R being CH₃) instead ofhydrogen, is connected to a heterocyclic group, typically a symmetricpyrimidine or triazine ring, having one or two substituents such asmethyl, ethyl, trifluoromethyl, methoxy, ethoxy, methylamino,dimethylamino, ethylamino and the halogens. Insulfonylaminocarbonyltriazolinone herbicides, the sulfonamide moiety isa component is a sulfonylaminocarbonyl bridge (—S(O)₂NHC(O)—). Insulfonylamino-carbonyltriazolinone herbicides the sulfonyl end of thesulfonylaminocarbonyl bridge is typically connected to substitutedphenyl ring. At the opposite end of the sulfonylaminocarbonyl bridge,the carbonyl is connected to the 1-position of a triazolinone ring,which is typically substituted with groups such as alkyl and alkoxy. Intriazolopyrimidine herbicides the sulfonyl end of the sulfonamide moietyis connected to the 2-position of a substituted[1,2,4]triazolopyrimidine ring system and the amino end of thesulfonamide moiety is connected to a substituted aryl, typically phenyl,group or alternatively the amino end of the sulfonamide moiety isconnected to the 2-position of a substituted [1,2,4]triazolopyrimidinering system and the sulfonyl end of the sulfonamide moiety is connectedto a substituted aryl, typically pyridinyl, group.

Representative of the sulfonylurea herbicides useful in the presentinvention are those of the formula:

wherein:

J is selected from the group consisting of

-   -   J is R¹³SO₂N(CH₃)—;    -   R is H or CH₃;    -   R¹ is F, Cl, Br, NO₂, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₃-C₄        cycloalkyl, C₂-C₄ haloalkenyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy,        C₂-C₄ alkoxyalkoxy, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰, CH₂CN or L;    -   R² is H, F, Cl, Br, I, CN, CH₃, OCH₃, SCH₃, CF₃ or OCF₂H;    -   R³ is Cl, NO₂, CO₂CH₃, CO₂CH₂CH₃, C(O)CH₃, C(O)CH₂CH₃,        C(O)-cyclopropyl, SO₂N(CH₃)₂, SO₂CH₃, SO₂CH₂CH₃, OCH₃ or        OCH₂CH₃;    -   R⁴ is C₁-C₃ alkyl, C₁-C₂ haloalkyl, C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, NO₂, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R⁵ is H, F, Cl, Br or CH₃;    -   R⁶ is C₁-C₃ alkyl optionally substituted with 0-3 F, 0-1 Cl and        0-1 C₃-C₄ alkoxyacetyloxy, or R⁶ is C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸, S(O),        R¹⁹, C(O)R²⁰ or L;    -   R⁷ is H, F, Cl, CH₃ or CF₃;    -   R⁸ is H, C₁-C₃ alkyl or pyridinyl;    -   R⁹ is C₁-C₃ alkyl, C₁-C₂ alkoxy, F, Cl, Br, NO₂, CO₂R¹⁴,        SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, OCF₂H, C(O)R²⁰, C₂-C₄ haloalkenyl or L;    -   R¹⁰ is H, Cl, F, Br, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹¹ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, C₂-C₄ haloalkenyl, F, Cl,        Br, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R¹² is halogen, C₁-C₄ alkyl or C₁-C₃ alkylsulfonyl;    -   R¹³ is C₁-C₄ alkyl;    -   R¹⁴ is allyl, propargyl or oxetan-3-yl; or R¹⁴ is C₁-C₃ alkyl        optionally substituted by at least one member independently        selected from halogen, C₁-C₂ alkoxy and CN;    -   R¹⁵ is H, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹⁶ is C₁-C₂ alkyl;    -   R¹⁷ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, allyl or cyclopropyl;    -   R¹⁸ is H or C₁-C₃ alkyl;    -   R¹⁹ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, allyl or propargyl;    -   R²⁰ is C₁-C₄ alkyl, C₁-C₄ haloalkyl or C₃-C₅ cycloalkyl        optionally substituted by halogen;    -   n is 0, 1 or 2;    -   L is    -   L¹ is CH₂, NH or O;    -   R²¹ is H or C₁-C₃ alkyl;    -   X is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        haloalkyl, C₁-C₄ haloalkylthio, C₁-C₄ alkylthio, halogen, C₂-C₅        alkoxyalkyl, C₂-C₅ alkoxyalkoxy, amino, C₁-C₃ alkylamino or        di(C₁-C₃ alkyl)amino;    -   Y is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        alkylthio, C₁-C₄ haloalkylthio, C₂-C₅ alkoxyalkyl, C₂-C₅        alkoxyalkoxy, amino, C₁-C₃ alkylamino, di(C₁-C₃ alkyl)amino,        C₃-C₄ alkenyloxy, C₃-C₄ alkynyloxy, C₂-C₅ alkylthioalkyl, C₂-C₅        alkylsulfinylalkyl, C₂-C₅ alkylsulfonylalkyl, C₁-C₄ haloalkyl,        C₂-C₄ alkynyl, C₃-C₅ cycloalkyl, azido or cyano; and    -   Z is CH or N;

provided that (i) when one or both of X and Y is C₁ haloalkoxy, then Zis CH; and (ii) when X is halogen, then Z is CH and Y is OCH₃, OCH₂CH₃,N(OCH₃)CH₃, NHCH₃, N(CH₃)₂ or OCF₂H. Of note is the present singleliquid herbicide composition comprising one or more sulfonylureas ofFormula I wherein when R⁶ is alkyl, said alkyl is unsubstituted.

Representative of the triazolopyrimidine herbicides contemplated for usein this invention are those of the formula:

wherein:

-   -   R²² and R²³ each independently halogen, nitro, C₁-C₄ alkyl,        C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy or C₂-C₃        alkoxycarbonyl;    -   R²⁴ is H, halogen, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   W is —NHS(O)₂— or —S(O)₂NH—;    -   Y¹ is H, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y² is H, F, Cl, Br, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y³ is H, F or methoxy;    -   Z¹ is CH or N; and    -   Z² is CH or N;        provided that at least one of Y¹ and Y² is other than H.

In the above Markush description of representative triazolopyrimidineherbicides, when W is —NHS(O)₂— the sulfonyl end of the sulfonamidemoiety is connected to the [1,2,4]triazolopyrimidine ring system, andwhen W is —S(O)₂NH— the amino end of the sulfonamide moiety is connectedto the [1,2,4]triazolopyrimidine ring system.

In the above recitations, the term “alkyl”, used either alone or incompound words such as “alkylthio” or “haloalkyl” includesstraight-chain or branched alkyl, such as, methyl, ethyl, n-propyl,i-propyl, or the different butyl isomers. “Cycloalkyl” includes, forexample, cyclopropyl, cyclobutyl and cyclopentyl. “Alkenyl” includesstraight-chain or branched alkenes such as ethenyl, 1-propenyl,2-propenyl, and the different butenyl isomers. “Alkenyl” also includespolyenes such as 1,2-propadienyl and 2,4-butadienyl. “Alkynyl” includesstraight-chain or branched alkynes such as ethynyl, 1-propynyl,2-propynyl and the different butynyl isomers. “Alkynyl” can also includemoieties comprised of multiple triple bonds such as 2,5-hexadiynyl.“Alkoxy” includes, for example, methoxy, ethoxy, n-propyloxy,isopropyloxy and the different butoxy isomers. “Alkoxyalkyl” denotesalkoxy substitution on alkyl. Examples of “alkoxyalkyl” include CH₃OCH₂,CH₃OCH₂CH₂, CH₃CH₂OCH₂, CH₃CH₂CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.“Alkoxyalkoxy” denotes alkoxy substitution on alkoxy. “Alkenyloxy”includes straight-chain or branched alkenyloxy moieties. Examples of“alkenyloxy” include H₂C═CHCH₂O, (CH₃)CH═CHCH₂O and CH₂=CHCH₂CH₂O.“Alkynyloxy” includes straight-chain or branched alkynyloxy moieties.Examples of “alkynyloxy” include HC≡CCH₂O and CH₃C≡CCH₂O. “Alkylthio”includes branched or straight-chain alkylthio moieties such asmethylthio, ethylthio, and the different propylthio isomers.“Alkylthioalkyl” denotes alkylthio substitution on alkyl. Examples of“alkylthioalkyl” include CH₃SCH₂, CH₃SCH₂CH₂, CH₃CH₂SCH₂,CH₃CH₂CH₂CH₂SCH₂ and CH₃CH₂SCH₂CH₂; “alkylsulfinylalkyl” and“alkylsulfonyl-alkyl” include the corresponding sulfoxides and sulfones,respectively. Other substituents such as “alkylamino”, “dialkylamino”are defined analogously.

The total number of carbon atoms in a substituent group is indicated bythe “C_(i)-C_(j)” prefix where i and j are numbers from 1 to 5. Forexample, C₁-C₄ alkyl designates methyl through butyl, including thevarious isomers. As further examples, C₂ alkoxyalkyl designates CH₃OCH₂;C₃ alkoxyalkyl designates, for example, CH₃CH(OCH₃), CH₃OCH₂CH₂ orCH₃CH₂OCH₂; and C₄ alkoxyalkyl designates the various isomers of analkyl group substituted with an alkoxy group containing a total of fourcarbon atoms, examples including CH₃CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.

The term “halogen”, either alone or in compound words such as“haloalkyl”, includes fluorine, chlorine, bromine or iodine. Further,when used in compound words such as “haloalkyl”, said alkyl may bepartially or fully substituted with halogen atoms which may be the sameor different. Examples of “haloalkyl” include F₃C, ClCH₂, CF₃CH₂ andCF₃CCl₂. The terms “haloalkoxy”, “haloalkylthio”, and the like, aredefined analogously to the term “haloalkyl”. Examples of “haloalkoxy”include CF₃O, CCl₃CH₂O, HCF₂CH₂CH₂O and CF₃CH₂O. Examples of“haloalkylthio” include CCl₃S, CF₃S, CCl₃CH₂S and ClCH₂CH₂CH₂S.

The following sulfonylurea herbicides illustrate the sulfonylureasuseful for this invention: amidosulfuron(N-[[[[(4,6-dimethoxy-2-pyrimdinyl)amino]carbonyl]amino]-sulfonyl]-N-methylmethanesulfonamide),azimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazol-5-yl)-1H-pyrazole-5-sulfonamide),bensulfuron-methyl (methyl2-[[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]methyl]benzoate),chlorimuron-ethyl (ethyl2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-benzoate),chlorsulfuron(2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]-carbonyl]benzenesulfonamide),cinosulfuron(N-[[(4,6-dimethoxy-1,3,5-triazin-2-yl)amino]carbonyl]-2-(2-methoxyethoxy)benzenesulfonamide),cyclosulfamuron(N-[[[2-(cyclopropylcarbonyl)phenyl]amino]sulfonyl]-N¹-(4,6-dimethoxypyrimidin-2-yl)urea),ethametsulfuron-methyl (methyl2-[[[[[4-ethoxy-6-(methylamino)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]benzoate),ethoxysulfuron (2-ethoxyphenyl[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]sulfamate), flazasulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(trifluoromethyl)-2-pyridinesulfonamide),flucetosulfuron(1-[3-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-2-pyridinyl]-2-fluoropropylmethoxyacetate), flupyrsulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyllamino]sulfonyl]-6-(trifluoromethyl)-3-pyridinecarboxylate),foramsulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-4-(formylamino)-N,N-dimethylbenzamide),halosulfuron-methyl(methyl3-chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),imazosulfuron(2-chloro-N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]imidazo[1,2-a]pyridine-3-sulfonamide),iodosulfuron-methyl (methyl4-iodo-2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),mesosulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-4-[[(methylsulfonyl)-amino]methyl]benzoate),metsulfuron-methyl (methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),nicosulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide),oxasulfuron (3-oxetanyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]benzoate),primisulfuron-methyl (methyl2-[[[[[4,6-bis(trifluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl]benzoate),prosulfuron(N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]-2-(3,3,3-trifluoropropyl)benzenesulfonamide),pyrazosulfuron-ethyl (ethyl5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),rimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide),sulfometuron-methyl(methyl-2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-benzoate),sulfosulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2-(ethylsulfonyl)imidazo[1,2-a]pyridine-3-sulfonamide),thifensulfuron-methyl (methyl3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate),triasulfuron(2-(2-chloroethoxy)-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzenesulfonamide),tribenuron-methyl (methyl2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylamino]carbonyl]-amino]sulfonyl]benzoate),trifloxysulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]-3-(2,2,2-trifluoroethoxy)-2-pyridinesulfonamide),triflusulfuron-methyl (methyl2-[[[[[4-dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]-3-methylbenzoate)and tritosulfuron(N-[[[4-methoxy-6-(trifluoromethyl)-1,3,5-triazin-2-yl]amino]carbonyl]-2-(trifluoromethyl)benzene-sulfonamide).

The following triazolopyrimidine herbicides illustrate thetriazolopyrimidines useful for this invention: cloransulam-methyl(methyl 3-chloro-2-[[(5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)sulfonyl]amino]benzoate, diclosulam(N-(2,6-dichlorophenyl)-5-ethoxy-7-fluoro[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide,florasulam(N-(2,6-difluorophenyl)-8-fluoro-5-methoxy[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide),flumetsulam(N-(2,6-difluorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),metosulam(N-(2,6-dichloro-3-methylphenyl)-5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),penoxsulam(2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)-6-(trifluoromethyl)benzenesulfonamide)and pyroxsulam(N-(5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy-4-(trifluoromethyl)-3-pyridinesulfonamide).

The following sulfonylaminocarbonyltriazolinone herbicides illustratethe sulfonylaminocarbonyltriazolinones useful for this invention:flucarbazone(4,5-dihydro-3-methoxy-4-methyl-5-oxo-N-[[2-(trifluoromethoxy)phenyl]sulfonyl]-1H-1,2,4-triazole-1-carboxamide)and procarbazone (methyl2-[[[(4,5-dihydro-4-methyl-5-oxo-3-propoxy-1H-1,2,4-triazol-1-yl)carbonyl]amino]sulfonyl]benzoate).

Additional herbicides include phenmedipham, triazolinones, and theherbicides disclosed in WO2006/012981, herein incorporated by referencein its entirety.

The methods further comprise applying to the crop and the weeds in thefield a sufficient amount of at least one herbicide to which the cropseeds or plants is tolerant, such as, for example, glyphosate, ahydroxyphenylpyruvatedioxygenase inhibitor (e.g., mesotrione orsulcotrione), a phytoene desaturase inhibitor (e.g., diflufenican), apigment synthesis inhibitor, sulfonamide, imidazolinone, bialaphos,phosphinothricin, azafenidin, butafenacil, sulfosate, glufosinate,triazolopyrimidine, pyrimidinyloxy(thio)benzoate, orsulonylaminocarbonyltriazolinone, an acetyl Co-A carboxylase inhibitorsuch as quizalofop-P-ethyl, a synthetic auxin such as quinclorac, or aprotox inhibitor to control the weeds without significantly damaging thecrop plants.

b. Effective Amount of a Herbicide

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. “Weed” as used herein refers to a plant which is notdesirable in a particular area. Conversely, a “crop plant” as usedherein refers to a plant which is desired in a particular area, such as,for example, a soybean plant. Thus, in some embodiments, a weed is anon-crop plant or a non-crop species, while in some embodiments, a weedis a crop species which is sought to be eliminated from a particulararea, such as, for example, an inferior and/or non-transgenic maizeplant in a field planted with transgenic maize, or a soybean plant in afield planted with corn. Weeds can be either classified into two majorgroups: monocots and dicots.

Many plant species can be controlled (i.e., killed or damaged) by theherbicides described herein. Accordingly, the methods of the inventionare useful in controlling these plant species where they are undesirable(i.e., where they are weeds). These plant species include crop plants aswell as species commonly considered weeds, including but not limited tospecies such as: blackgrass (Alopecurus myosuroides), giant foxtail(Setaria faberi), large crabgrass (Digitaria sanguinalis), Surinam grass(Brachiaria decumbens), wild oat (Avena fatua), common cocklebur(Xanthium pensylvanicum), common lambsquarters (Chenopodium album),morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf(Abutilion theophrasti), common barnyardgrass (Echinochloa crus-galli),bermudagrass (Cynodon dactylon), downy brome (Bromus tectorum),goosegrass (Eleusine indica), green foxtail (Setaria viridis), Italianryegrass (Lolium multiflorum), Johnsongrass (Sorghum halepense), lessercanarygrass (Phalaris minor), windgrass (Apera spica-venti), woolycupgrass (Erichloa villosa), yellow nutsedge (Cyperus esculentus),common chickweed (Stellaria media), common ragweed (Ambrosiaartemisuifolia), Kochia scoparia, horseweed (Conyza canadensis), rigidryegrass (Lolium rigidum), goosegrass (Eleucine indica), hairy fleabane(Conyza bonariensis), buckhorn plantain (Plantago lanceolata), tropicalspiderwort (Commelina benghalensis), field bindweed (Convolvulusarvensis), purple nutsedge (Cyperus rotundus), redvine (Brunnichiaovata), hemp sesbania (Sesbania exaltata), sicklepod (Sennaobtusifolia), Texas blueweed (Helianthus ciliaris), and Devil's claws(Proboscidea louisianica). In other embodiments, the weed comprises aherbicide-resistant ryegrass, for example, a glyphosate resistantryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistantryegrass, and a non-selective herbicide resistant ryegrass. In someembodiments, the undesired plants are proximate the crop plants.

As used herein, by “selectively controlled” is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cropplants are significantly damaged or killed.

In some embodiments, a glyphosate/ALS inhibitor-tolerant plant of theinvention is not significantly damaged by treatment with a particularherbicide applied to that plant at a dose equivalent to a rate of atleast 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,150, 170, 200, 300, 400, 500, 600, 700, 800, 800, 1000, 2000, 3000,4000, 5000 or more grams or ounces (1 ounce=29.57 ml) of activeingredient or commercial product or herbicide formulation per acre orper hectare, whereas an appropriate control plant is significantlydamaged by the same treatment.

In specific embodiments, an effective amount of an ALS inhibitorherbicide comprises at least about 0.1, 1, 5, 10, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 850, 900, 950,1000, 2000, 3000, 4000, 5000, or more grams or ounces (1 ounce=29.57 ml)of active ingredient per hectare. In other embodiments, an effectiveamount of an ALS inhibitor comprises at least about 0.1-50, about 25-75,about 50-100, about 100-110, about 110-120, about 120-130, about130-140, about 140-150, about 150-200, about 200-500, about 500-600,about 600-800, about 800-1000, or greater grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Any ALS inhibitor, for example,those listed in Table 1 can be applied at these levels.

In other embodiments, an effective amount of a sulfonylurea comprises atleast 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 5000 or more grams or ounces (1ounce=29.57 ml) of active ingredient per hectare. In other embodiments,an effective amount of a sulfonylurea comprises at least about 0.1-50,about 25-75, about 50-100, about 100-110, about 110-120, about 120-130,about 130-140, about 140-150, about 150-160, about 160-170, about170-180, about 190-200, about 200-250, about 250-300, about 300-350,about 350-400, about 400-450, about 450-500, about 500-550, about550-600, about 600-650, about 650-700, about 700-800, about 800-900,about 900-1000, about 1000-2000, or more grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Representative sulfonylureas thatcan be applied at this level are set forth in Table 1.

In other embodiments, an effective amount of asulfonylaminocarbonyltriazolinones, triazolopyrimidines,pyrimidinyloxy(thio)benzoates, and imidazolinones can comprise at leastabout 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1500, 1550, 1600, 1650, 1700, 1800,1850, 1900, 1950, 2000, 2500, 3500, 4000, 4500, 5000 or greater grams orounces (1 ounce=29.57 ml) active ingredient per hectare. In otherembodiments, an effective amount of a sulfonyluminocarbonyltriazolines,triazolopyrimidines, pyrimidinyloxy(thio)benzoates, or imidazolinonescomprises at least about 0.1-50, about 25-75, about 50-100, about100-110, about 110-120, about 120-130, about 130-140, about 140-150,about 150-160, about 160-170, about 170-180, about 190-200, about200-250, about 250-300, about 300-350, about 350-400, about 400-450,about 450-500, about 500-550, about 550-600, about 600-650, about650-700, about 700-800, about 800-900, about 900-1000, about 1000-2000,or more grams or ounces (1 ounce=29.57 ml) active ingredient perhectare.

Additional ranges of the effective amounts of herbicides can be found,for example, in various publications from University Extension services.See, for example, Bernards et al. (2006) Guide for Weed Management inNebraska (www.ianrpubs.url.edu/sendlt/ec130); Regher et al. (2005)Chemical Weed Control for Fields Crops, Pastures, Rangeland, andNoncropland, Kansas State University Agricultural Extension Station andCorporate Extension Service; Zollinger et al. (2006) North Dakota WeedControl Guide, North Dakota Extension Service, and the Iowa StateUniversity Extension at www.weeds.iastate.edu, each of which is hereinincorporated by reference.

In some embodiments of the invention, glyphosate is applied to an areaof cultivation and/or to at least one plant in an area of cultivation atrates between 8 and 32 ounces of acid equivalent per acre, or at ratesbetween 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 ounces of acidequivalent per acre at the lower end of the range of application andbetween 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 ounces of acidequivalent per acre at the higher end of the range of application (1ounce=29.57 ml). In other embodiments, glyphosate is applied at least at1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater ounce of activeingredient per hectare (1 ounce=29.57 ml). In some embodiments of theinvention, a sulfonylurea herbicide is applied to a field and/or to atleast one plant in a field at rates between 0.04 and 1.0 ounces ofactive ingredient per acre, or at rates between 0.1, 0.2, 0.4, 0.6, and0.8 ounces of active ingredient per acre at the lower end of the rangeof application and between 0.2, 0.4, 0.6, 0.8, and 1.0 ounces of activeingredient per acre at the higher end of the range of application. (1ounce=29.57 ml)

As is known in the art, glyphosate herbicides as a class contain thesame active ingredient, but the active ingredient is present as one of anumber of different salts and/or formulations. However, herbicides knownto inhibit ALS vary in their active ingredient as well as their chemicalformulations. One of skill in the art is familiar with the determinationof the amount of active ingredient and/or acid equivalent present in aparticular volume and/or weight of herbicide preparation.

In some embodiments, an ALS inhibitor herbicide is employed. Rates atwhich the ALS inhibitor herbicide is applied to the crop, crop part,seed or area of cultivation can be any of the rates disclosed herein. Inspecific embodiments, the rate for the ALS inhibitor herbicide is about0.1 to about 5000 g ai/hectare, about 0.5 to about 300 g ai/hectare, orabout 1 to about 150 g ai/hectare.

Generally, a particular herbicide is applied to a particular field (andany plants growing in it) no more than 1, 2, 3, 4, 5, 6, 7, or 8 times ayear, or no more than 1, 2, 3, 4, or 5 times per growing season.

By “treated with a combination of” or “applying a combination of”herbicides to a crop, area of cultivation or field” is intended that aparticular field, crop or weed is treated with each of the herbicidesand/or chemicals indicated to be part of the combination so that desiredeffect is achieved, i.e., so that weeds are selectively controlled whilethe crop is not significantly damaged. In some embodiments, weeds whichare susceptible to each of the herbicides exhibit damage from treatmentwith each of the herbicides which is additive or synergistic. Theapplication of each herbicide and/or chemical may be simultaneous or theapplications may be at different times, so long as the desired effect isachieved. Furthermore, the application can occur prior to the plantingof the crop.

The proportions of herbicides used in the methods of the invention withother herbicidal active ingredients in herbicidal compositions aregenerally in the ratio of 5000:1 to 1:5000, 1000:1 to 1:1000, 100:1 to1:100, 10:1 to 1:10 or 5:1 to 1:5 by weight. The optimum ratios can beeasily determined by those skilled in the art based on the weed controlspectrum desired. Moreover, any combinations of ranges of the variousherbicides disclosed in Table 3 can also be applied in the methods ofthe invention.

Thus, in some embodiments, the invention provides improved methods forselectively controlling weeds in a field wherein the total herbicideapplication may be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of that used inother methods. Similarly, in some embodiments, the amount of aparticular herbicide used for selectively controlling weeds in a fieldmay be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the amount of that particularherbicide that would be used in other methods, i.e., methods notutilizing a plant of the invention.

In some embodiments, a glyphosate/ALS inhibitor-tolerant plant of theinvention benefits from a synergistic effect wherein the herbicidetolerance conferred by a polypeptide that confers resistance toglyphosate (i.e., GAT) and at least an ALS inhibitor-tolerantpolypeptide is greater than expected from simply combining the herbicidetolerance conferred by each gene separately to a transgenic plantcontaining them individually. See, e.g., McCutchen et al. (1997) J.Econ. Entomol. 90: 1170-1180; Priesler et al. (1999) J. Econ. Entomol.92: 598-603. As used herein, the terms “synergy,” “synergistic,”“synergistically” and derivations thereof, such as in a “synergisticeffect” or a “synergistic herbicide combination” or a “synergisticherbicide composition” refer to circumstances under which the biologicalactivity of a combination of herbicides, such as at least a firstherbicide and a second herbicide, is greater than the sum of thebiological activities of the individual herbicides. Synergy, expressedin terms of a “Synergy Index (SI),” generally can be determined by themethod described by F. C. Kull, et al., Applied Microbiology 9, 538(1961). See also Colby S. R., “Calculating Synergistic and AntagonisticResponses of Herbicide Combinations,” Weeds 15, 20-22 (1967).

In other instances, the herbicide tolerance conferred on aglyphosate/ALS inhibitor-tolerant plant of the invention is additive;that is, the herbicide tolerance profile conferred by the herbicidetolerance genes is what would be expected from simply combining theherbicide tolerance conferred by each gene separately to a transgenicplant containing them individually. Additive and/or synergistic activityfor two or more herbicides against key weed species will increase theoverall effectiveness and/or reduce the actual amount of activeingredient(s) needed to control said weeds. Where such synergy isobserved, the plant of the invention may display tolerance to a higherdose or rate of herbicide and/or the plant may display tolerance toadditional herbicides or other chemicals beyond those to which it wouldbe expected to display tolerance. For example, a plant containing a GATgene and an HRA gene may show tolerance to organophosphate compoundssuch as insecticides and/or inhibitors of 4-hydroxyphenylpyruvatedioxygenase.

Thus, for example, glyphosate/ALS inhibitor-tolerant plants of theinvention can exhibit greater than expected tolerance to variousherbicides, including but not limited to glyphosate, ALS inhibitorchemistries, and sulfonylurea herbicides. The glyphosate/ALSinhibitor-tolerant plants of the invention may show tolerance to aparticular herbicide or herbicide combination that is at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%, 150%,175%, 200%, 300%, 400%, or 500% or more higher than the tolerance of anappropriate control plant that contains only a single herbicidetolerance gene which confers tolerance to the same herbicide orherbicide combination. Thus, glyphosate/ALS inhibitor-tolerant plants ofthe invention may show decreased damage from the same dose of herbicidein comparison to an appropriate control plant, or they may show the samedegree of damage in response to a much higher dose of herbicide than thecontrol plant. Accordingly, in specific embodiments, a particularherbicide used for selectively containing weeds in a field is more than1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%,100% or greater than the amount of that particular herbicide that wouldbe used in other methods, i.e., methods not utilizing a plant of theinvention.

In the same manner, in some embodiments, a glyphosate/ALSinhibitor-tolerant plant of the invention shows improved tolerance to aparticular formulation of a herbicide active ingredient in comparison toan appropriate control plant. Herbicides are sold commercially asformulations which typically include other ingredients in addition tothe herbicide active ingredient; these ingredients are often intended toenhance the efficacy of the active ingredient. Such other ingredientscan include, for example, safeners and adjuvants (see, e.g., Green andFoy (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. Inderjit (Kluwer Academic Publishers,The Netherlands)). Thus, a glyphosate/ALS inhibitor-tolerant plant ofthe invention can show tolerance to a particular formulation of aherbicide (e.g., a particular commercially available herbicide product)that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%,17%,20%, 22%, 25%, 27%,30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, 1100%, 1200%, 1300%,1400%, 1500%, 1600%, 1700%, 1800%,1900%, or 2000% or more higher than the tolerance of an appropriatecontrol plant that contains only a single herbicide tolerance gene whichconfers tolerance to the same herbicide formulation.

In some embodiments, a glyphosate/ALS inhibitor-tolerant plant of theinvention shows improved tolerance to a herbicide or herbicide class towhich the at least one other herbicide tolerance gene confers toleranceas well as improved tolerance to at least one other herbicide orchemical which has a different mechanism or basis of action than eitherglyphosate or the herbicide corresponding to said at least one otherherbicide tolerance gene. This surprising benefit of the invention findsuse in methods of growing crops that comprise treatment with variouscombinations of chemicals, including, for example, other chemicals usedfor growing crops. Thus, for example, a glyphosate/ALSinhibitor-tolerant maize plant of the invention (i.e., a GAT/HRA plant)may also show improved tolerance to chlorpyrifos, a systemicorganophosphate insecticide which interferes with the ability of maizeto metabolize herbicide via interference with the cytochrome P450 gene.Thus, the invention also provides a transgenic plant comprising asequence that confers tolerance to glyphosate (i.e., a GAT gene) and asulfonylurea herbicide tolerance gene which shows improved tolerance tochemicals which affect the cytochrome P450 gene, and methods of usethereof. In some embodiments, glyphosate/ALS inhibitor-tolerant plantsof the invention comprising, for example, a GAT gene and a sulfonylureaherbicide tolerance gene also show improved tolerance to dicamba. Inthese embodiments, the improved tolerance to dicamba may be evident inthe presence of glyphosate and a sulfonylurea herbicide.

In other methods, a herbicide combination is applied over aglyphosate/ALS inhibitor-tolerant plant of the invention, where theherbicide combination produces either an additive or a synergisticeffect for controlling weeds. Such combinations of herbicides can allowthe application rate to be reduced, a broader spectrum of undesiredvegetation to be controlled, improved control of the undesiredvegetation with fewer applications, more rapid onset of the herbicidalactivity, or more prolonged herbicidal activity.

An “additive herbicidal composition” has a herbicidal activity that isabout equal to the observed activities of the individual components. A“synergistic herbicidal combination” has a herbicidal activity higherthan what can be expected based on the observed activities of theindividual components when used alone. Accordingly, the presentlydisclosed subject matter provides a synergistic herbicide combination,wherein the degree of weed control of the mixture exceeds the sum ofcontrol of the individual herbicides. In some embodiments, the degree ofweed control of the mixture exceeds the sum of control of the individualherbicides by any statistically significant amount including, forexample, about 1% to 5%, about 5% to about 10%, about 10% to about 20%,about 20% to about 30%, about 30% to 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 100%, about 100% to 120% orgreater. Further, a “synergistically effective amount” of a herbiciderefers to the amount of one herbicide necessary to elicit a synergisticeffect in another herbicide present in the herbicide composition. Thus,the term “synergist,” and derivations thereof, refer to a substance thatenhances the activity of an active ingredient (ai), i.e., a substance ina formulation from which a biological effect is obtained, for example, aherbicide.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for controlling weeds in an area of cultivation. Insome embodiments, the method comprises: (a) planting the area with cropseeds or crop plants, wherein the crop seeds or crop plants comprise:(i) a first polynucleotide encoding a polypeptide that can confertolerance to glyphosate operably linked to a promoter active in the cropseeds or crop plants; and (ii) a second polynucleotide encoding an ALSinhibitor-tolerant polypeptide operable linked to a promoter active inthe crop seeds or crop plants; and (b) applying to the weed, the cropplants, a crop part, the area of cultivation, or a combination thereof,an effective amount of a herbicide composition comprising at least oneof a synergistically effective amount of glyphosate and asynergistically effective amount of an ALS inhibitor (for example, butnot limited to, a sulfonylurea herbicide), or agriculturally suitablesalts thereof, wherein at least one of: (i) the synergisticallyeffective amount of the glyphosate is lower than an amount of glyphosaterequired to control the weeds in the absence of the sulfonylureaherbicide; (ii) the synergistically effective amount of the ALSinhibitor herbicide is lower than an amount of the ALS inhibitorrequired-to control the weeds in the absence of glyphosate; and (iii)combinations thereof; and wherein the effective amount of the herbicidecomposition is tolerated by the crop seeds or crop plants and controlsthe weeds in the area of cultivation.

As described in more detail hereinabove, in some embodiments, the firstpolynucleotide encodes a glyphosate-N-acetyltransferase. Moreparticularly, in some embodiments, the first polynucleotide encodes aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase or aglyphosate-tolerant glyphosate oxido-reductase. Further, as alsodescribed in more detail hereinabove, the ALS inhibitor-tolerantpolypeptide comprises a mutated acetolactate synthase polypeptide. Insome embodiments, the mutated acetolactate synthase polypeptidecomprises HRA.

In some embodiments, the herbicide composition used in the presentlydisclosed method for controlling weeds comprises a synergisticallyeffective amount of glyphosate and a sulfonylurea herbicide. In furtherembodiments, the presently disclosed synergistic herbicide compositioncomprises glyphosate and a sulfonylurea herbicide selected from thegroup consisting of metsulfuron-methyl, chlorsulfuron, and triasulfuron.

In particular embodiments, the synergistic herbicide combination furthercomprises an adjuvant such as, for example, an ammonium sulfate-basedadjuvant, e.g., ADD-UP® (Wenkem S. A., Halfway House, Midrand, SouthAfrica). In additional embodiments, the presently disclosed synergisticherbicide compositions comprise an additional herbicide, for example, aneffective amount of a pyrimidinyloxy(thio)benzoate herbicide. In someembodiments, the pyrimidinyloxy(thio)benzoate herbicide comprisesbispyribac, e.g., (VELOCITY®, Valent U.S.A. Corp., Walnut Creek, Calif.,United States of America), or an agriculturally suitable salt thereof.

In some embodiments of the presently disclosed method for controllingundesired plants, the glyphosate is applied pre-emergence,post-emergence or pre- and post-emergence to the undesired plants orplant crops; and the ALS inhibitor herbicide (i.e., the sulfonylureaherbicide) is applied pre-emergence, post-emergence or pre- andpost-emergence to the undesired plants or plant crops. In otherembodiments, the glyphosate and the ALS inhibitor herbicide (i.e., thesulfonylurea herbicide) are applied together or are applied separately.In yet other embodiments, the synergistic herbicide composition isapplied, e.g. step (b) above, at least once prior to planting thecrop(s) of interest, e.g., step (a) above.

While the glyphosate/ALS inhibitor-tolerant plants of the invention aretolerant to many herbicides, they are not tolerant to severalherbicides, such as, for example, dinitroaniline, ACCase, andchloracetamide herbicides. Thus, methods of the invention that comprisethe control of weeds may also make use of these treatments to controlglyphosate/ALS inhibitor-tolerant plants, such as, for example,“volunteer” glyphosate/ALS inhibitor-tolerant plants crops that arise ina field that has been planted or replanted with a different crop.

Weeds that can be difficult to control with glyphosate alone in fieldswhere a crop is grown (such as, for example, a soybean crop) include butare not limited to the following: horseweed (e.g., Conyza canadensis);rigid ryegrass (e.g., Lolium rigidum); goosegrass (e.g., Eleusineindica); Italian ryegrass (e.g., Lolium multiflorum); hairy fleabane(e.g., Conyza bonariensis); buckhorn plantain (e.g., Plantagolanceolata); common ragweed (e.g., Ambrosia artemisifolia); morningglory (e.g., Ipomoea spp.); waterhemp (e.g., Amaranthus spp.); fieldbindweed (e.g., Convolvulus arvensis); yellow nutsedge (e.g., Cyperusesculentus); common lambsquarters (e.g., Chenopodium album); wildbuckwheat (e.g., Polygonium convolvulus); velvetleaf (e.g., Abutilontheophrasti); kochia (e.g., Kochia scoparia); and Asiatic dayflower(e.g., Commelina spp.). In areas where such weeds are found, theglyphosate/ALS inhibitor-tolerant plants of the invention (GAT-HRAplants) are particularly useful in allowing the treatment of a field(and therefore any crop growing in the field) with combinations ofherbicides that would cause unacceptable damage to crop plants that didnot contain both of these polynucleotides. Plants of the invention thatare tolerant to glyphosate and other herbicides such as, for example,sulfonylurea, imidazolinone, triazolopyrimidine,pyrimidinyl(thio)benzoate, and/or sulfonylaminocarbonyltriazolinoneherbicides in addition to being tolerant to at least one other herbicidewith a different mode of action or site of action are particularlyuseful in situations where weeds are tolerant to at least two of thesame herbicides to which the plants are tolerant. In this manner, plantsof the invention make possible improved control of weeds that aretolerant to more than one herbicide.

For example, some commonly used treatments for weed control in fieldswhere current commercial crops (including, for example, soybeans) aregrown include glyphosate and, optionally, 2,4-D; this combination,however, has some disadvantages. Particularly, there are weed speciesthat it does not control well and it also does not work well for weedcontrol in cold weather. Another commonly used treatment for weedcontrol in soybean fields is the sulfonylurea herbicidechlorimuron-ethyl, which has significant residual activity in the soiland thus maintains selective pressure on all later-emerging weedspecies, creating a favorable environment for the growth and spread ofsulfonylurea-resistant weeds. However, the glyphosate/ALSinhibitor-tolerant plants (i.e., GAT-HRA plants of the invention),including glyphosate/ALS inhibitor-tolerant soybean plants (i.e.,GAT-HRA soybean plants), can be treated with herbicides (e.g.,chlorimuron-ethyl) and combinations of herbicides that would causeunacceptable damage to standard plant varieties. Thus, for example,fields containing the glyphosate/ALS inhibitor-tolerant soybean plant(i.e., GAT-HRA soybean plants) can be treated with sulfonylurea,imidazolinone, triazolopyrimidines, pyrimidiny(thio)benzoates, and/orsulfonylaminocarbonyltriazonlinone such as the sulfonylureachlorimuron-ethyl, either alone or in combination with other herbicides.For example, fields containing soybean plants of the invention can betreated with a combination of glyphosate and tribenuron-methyl(available commercially as Express®). This combination has severaladvantages for weed control under some circumstances, including the useof herbicides with different modes of action and the use of herbicideshaving a relatively short period of residual activity in the soil. Aherbicide having a relatively short period of residual activity isdesirable, for example, in situations where it is important to reduceselective pressure that would favor the growth of herbicide-tolerantweeds. Of course, in any particular situation where weed control isrequired, other considerations may be more important, such as, forexample, the need to prevent the development of and/or appearance ofweeds in a field prior to planting a crop by using a herbicide with arelatively long period of residual activity. The glyphosate/ALSinhibitor-tolerant soybean plants can also be treated with herbicidecombinations that include at least one of nicosulfuron,metsulfuron-methyl, tribenuron-methyl, thifensulfuron-methyl, and/orrimsulfuron. Treatments that include both tribenuron-methyl andthifensulfuron-methyl may be particularly useful.

Other commonly used treatments for weed control in fields where currentcommercial varieties of crops (including, for example, soybeans) aregrown include the sulfonylurea herbicide thifensulfuron-methyl(available commercially as Harmony GT®). However, one disadvantage ofthifensulfuron-methyl is that the higher application rates required forconsistent weed control often cause injury to a crop growing in the samefield. The glyphosate/ALS inhibitor-tolerant plants of the invention,including soybean plants, can be treated with a combination ofglyphosate and thifensulfuron-methyl, which has the advantage of usingherbicides with different modes of action. Thus, weeds that areresistant to either herbicide alone are controlled by the combination ofthe two herbicides, and the glyphosate/ALS inhibitor-tolerant plants ofthe invention are not significantly damaged by the treatment.

Other herbicides which are used for weed control in fields where currentcommercial varieties of crops (including, for example, soybeans) aregrown are the triazolopyrimidine herbicide cloransulam-methyl (availablecommercially as FirstRate®) and the imidazolinone herbicide imazaquin(available commercially as Sceptor®). When these herbicides are usedindividually they may provide only marginal control of weeds. However,fields containing the glyphosate/ALS inhibitor-tolerant plants of theinvention, including soybean plants, can be treated, for example, with acombination of glyphosate (e.g., Roundup® (glyphosate isopropylaminesalt)), imazapyr (currently available commercially as Arsenal®),chlorimuron-ethyl (currently available commercially as Classic®),quizalofop-P-ethyl (currently available commercially as Assure II®), andfomesafen (currently available commercially as Flexstar®). Thiscombination has the advantage of using herbicides with different modesof action. Thus, weeds that are tolerant to just one or several of theseherbicides are controlled by the combination of the five herbicides, andthe glyphosate/ALS inhibitor-tolerant plants of the invention are notsignificantly damaged by treatment with this herbicide combination. Thiscombination provides an extremely broad spectrum of protection againstthe type of herbicide-tolerant weeds that might be expected to arise andspread under current weed control practices.

Fields containing the glyphosate/ALS inhibitor-tolerant plants of theinvention (i.e., GAT/HRA plants), including soybean plants, may also betreated, for example, with a combination of herbicides includingglyphosate, rimsulfuron, and dicamba or mesotrione. This combination maybe particularly useful in controlling weeds which have developed sometolerance to herbicides which inhibit ALS. Another combination ofherbicides which may be particularly useful for weed control includesglyphosate and at least one of the following: metsulfuron-methyl(commercially available as Ally®), imazapyr (commercially available asArsenal®), imazethapyr, imazaquin, and sulfentrazone. It is understoodthat any of the combinations discussed above or elsewhere herein mayalso be used to treat areas in combination with any other herbicide oragricultural chemical.

Some commonly-used treatments for weed control in fields where currentcommercial crops (including, for example, maize) are grown includeglyphosate (currently available commercially as Roundup®), rimsulfuron(currently available commercially as Resolve® or Matrix®), dicamba(commercially available as Clarity®), atrazine, and mesotrione(commercially available as Callisto®). These herbicides are sometimesused individually due to poor crop tolerance to multiple herbicides.Unfortunately, when used individually, each of these herbicides hassignificant disadvantages. Particularly, the incidence of weeds that aretolerant to individual herbicides continues to increase, renderingglyphosate less effective than desired in some situations. Rimsulfuronprovides better weed control at high doses which can cause injury to acrop, and alternatives such as dicamba are often more expensive thanother commonly-used herbicides. However, glyphosate/ALSinhibitor-tolerant plants (i.e., GAT-HRA plants) of the invention,including glyphosate/ALS inhibitor-tolerant maize plants, can be treatedwith herbicides and combinations of herbicides that would causeunacceptable damage to standard plant varieties, including combinationsof herbicides that comprise rimsulfuron and/or dicamba. Other suitablecombinations of herbicides for use with glyphosate/ALSinhibitor-tolerant plants of the invention include glyphosate,sulfonylurea, imidazolinone, triazolopyrimidine,pryimidinyloxy(thio)benzoates, and/or sulfonylaminocarbonyltriazonlinoneherbicides, including, for example, and at least one of the following:metsulfuron-methyl, tribenuron-methyl, chlorimuron-ethyl, imazethapyr,imazapyr, and imazaquin.

For example, glyphosate/ALS inhibitor-tolerant maize plants (i.e.GAT/HRA plants) can be treated with a combination of glyphosate andrimsulfuron, or a combination or rimsulfuron and at least one otherherbicide. Glyphosate/ALS inhibitor plants (i.e., GAT-HRA plants) canalso be treated with a combination of glyphosate, rimsulfuron, anddicamba, or a combination of glyphosate, rimsulfuron, and at least oneother herbicide. In some embodiments, the at least one other herbicidehas a different mode of action than both glyphosate and rimsulfuron. Thecombination of glyphosate, rimsulfuron, and dicamba has the advantagethat these herbicides have different modes of action and short residualactivity, which decreases the risk of incidence and spread ofherbicide-tolerant weeds.

Some commonly-used treatments for weed control in fields where currentcommercial crops (including, for example, cotton) are grown includeglyphosate (currently available commercially as Roundup®),chlorimuron-ethyl, tribenuron-methyl, rimsulfuron (currently availablecommercially as Resolve® or Matrix®), imazethapyr, imazapyr, andimazaquin. Unfortunately, when used individually, each of theseherbicides has significant disadvantages. Particularly, the incidence ofweeds that are tolerant to individual herbicides continues to increase,rendering each individual herbicide less effective than desired in somesituations. However, glyphosate/ALS inhibitor-tolerant plants of theinvention, including cotton plants, can be treated with a combination ofherbicides that would cause unacceptable damage to standard plantvarieties, including combinations of herbicides that include at leastone of those mentioned above.

c. Methods of Herbicide Application

In the methods of the invention, a herbicide may be formulated andapplied to an area of interest such as, for example, a field or area ofcultivation, in any suitable manner. A herbicide may be applied to afield in any form, such as, for example, in a liquid spray or as solidpowder or granules. In specific embodiments, the herbicide orcombination of herbicides that are employed in the methods comprise atankmix or a premix. A herbicide may also be formulated, for example, asa “homogenous granule blend” produced using blends technology (see,e.g., U.S. Pat. No. 6,022,552, entitled “Uniform Mixtures of PesticideGranules”). The blends technology of U.S. Pat. No. 6,022,552 produces anonsegregating blend (i.e., a “homogenous granule blend”) of formulatedcrop protection chemicals in a dry granule form that enables delivery ofcustomized mixtures designed to solve specific problems. A homogenousgranule blend can be shipped, handled, subsampled, and applied in thesame manner as traditional premix products where multiple activeingredients are formulated into the same granule.

Briefly, a “homogenous granule blend” is prepared by mixing together atleast two extruded formulated granule products. In some embodiments,each granule product comprises a registered formulation containing asingle active ingredient which is, for example, a herbicide, afungicide, and/or an insecticide. The uniformity (homogeneity) of a“homogenous granule blend” can be optimized by controlling the relativesizes and size distributions of the granules used in the blend. Thediameter of extruded granules is controlled by the size of the holes inthe extruder die, and a centrifugal sifting process may be used toobtain a population of extruded granules with a desired lengthdistribution (see, e.g., U.S. Pat. No. 6,270,025).

A homogenous granule blend is considered to be “homogenous” when it canbe subsampled into appropriately sized aliquots and the composition ofeach aliquot will meet the required assay specifications. To demonstratehomogeneity, a large sample of the homogenous granule blend is preparedand is then subsampled into aliquots of greater than the minimumstatistical sample size (see Example 4).

In non-limiting embodiments, the glyphosate/ALS inhibitor-tolerant plant(i.e., a GAT-HRA plant), including a soybean plant, can be treated withherbicides (e.g., chlorimuron-ethyl and combinations of other herbicidesthat without the glyphosate/ALS inhibitor-tolerant crop would havecaused unacceptable crop response to plant varieties without theglyphosate/ALS inhibitor genetics). Thus, for example, fields plantedwith and containing glyphosate/ALS inhibitor-tolerant soybean, corn orcotton varieties (i.e., GAT/HRA plants) can be treated withsulfonylurea, imidazolinone, triazolopyrimidine,pyrimidinyl(thio)benzoate, and/or sulfonylaminocarbonyltriazonlinoneherbicides, either alone or in combination with other herbicides. SinceALS inhibitor chemistries have different herbicidal attributes, blendsof ALS plus other chemistries will provide superior weed managementstrategies including varying and increased weed spectrum, the ability toprovide specified residual activity (SU/ALS inhibitor chemistry withresidual activity leads to improved foliar activity which leads to awider window between glyphosate applications, as well as, an addedperiod of control if weather conditions prohibit timely application).

Blends also afford the ability to add other agrochemicals at normal,labeled use rates such as additional herbicides (a 3^(rd)/4^(th)mechanism of action), fungicides, insecticides, plant growth regulatorsand the like thereby saving costs associated with additionalapplications.

Any herbicide formulation applied over the glyphosate/ALSinhibitor-tolerant plant can be prepared as a “tank-mix” composition. Insuch embodiments, each ingredient or a combination of ingredients can bestored separately from one another. The ingredients can then be mixedwith one another prior to application. Typically, such mixing occursshortly before application. In a tank-mix process, each ingredient,before mixing, typically is present in water or a suitable organicsolvent. For additional guidance regarding the art of formulation, seeT. S. Woods, “The Formulator's Toolbox—Product Forms for ModernAgriculture” Pesticide Chemistry and Bioscience, The Food-EnvironmentChallenge, T. Brooks and T. R. Roberts, Eds., Proceedings of the 9thInternational Congress on Pesticide Chemistry, The Royal Society ofChemistry, Cambridge, 1999, pp. 120-133. See also U.S. Pat. No.3,235,361, Col. 6, line 16 through Col. 7, line 19 and Examples 10-41;U.S. Pat. No. 3,309,192, Col. 5, line 43 through Col. 7, line 62 andExamples 8, 12, 15, 39, 41, 52, 53, 58, 132, 138-140, 162-164, 166, 167and 169-182; U.S. Pat. No. 2,891,855, Col. 3, line 66 through Col. 5,line 17 and Examples 1-4; Klingman, Weed Control as a Science, JohnWiley and Sons, Inc., New York, 1961, pp 81-96; and Hance et al., WeedControl Handbook, 8th Ed., Blackwell Scientific Publications, Oxford,1989, each of which is incorporated herein by reference in theirentirety.

The methods of the invention further allow for the development ofherbicide combinations to be used in with the glyphosate/ALSinhibitor-tolerant plants. In such methods, the environmental conditionsin an area of cultivation are evaluated. Environmental conditions thatcan be evaluated include, but are not limited to, ground and surfacewater pollution concerns, intended use of the crop, crop tolerance, soilresiduals, weeds present in area of cultivation, soil texture, pH ofsoil, amount of organic matter in soil, application equipment, andtillage practices. Upon the evaluation of the environmental conditions,an effective amount of a combination of herbicides can be applied to thecrop, crop part, seed of the crop or area of cultivation.

d. Timing of Herbicide Application

In some embodiments, the herbicide applied to the glyphosate/ALSinhibitor-tolerant plants of the invention serves to prevent theinitiation of growth of susceptible weeds and/or serve to cause damageto weeds that are growing in the area of interest. In some embodiments,the herbicide or herbicide mixture exert these effects on weedsaffecting crops that are subsequently planted in the area of interest(i.e., field or area of cultivation). In the methods of the invention,the application of the herbicide combination need not occur at the sametime. So long as the field in which the crop is planted containsdetectable amounts of the first herbicide and the second herbicide isapplied at some time during the period in which the crop is in the areaof cultivation, the crop is considered to have been treated with amixture of herbicides according to the invention. Thus, methods of theinvention encompass applications of herbicide which are “preemergent,”“postemergent,” “preplant incorporation” and/or which involve seedtreatment prior to planting.

In one embodiment, methods are provided for coating seeds. The methodscomprise coating a seed with an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). The seeds canthen be planted in an area of cultivation. Further provided are seedshaving a coating comprising an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein).

“Preemergent” refers to a herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Postemergent” refers to a herbicide which isapplied to an area after a plant emerges visibly from the soil. In someinstances, the terms “preemergent” and “postemergent” are used withreference to a weed in an area of interest, and in some instances theseterms are used with reference to a crop plant in an area of interest.When used with reference to a weed, these terms may apply to only aparticular type of weed or species of weed that is present or believedto be present in the area of interest. While any herbicide may beapplied in a preemergent and/or postemergent treatment, some herbicidesare known to be more effective in controlling a weed or weeds whenapplied either preemergence or postemergence. For example, rimsulfuronhas both preemergence and postemergence activity, while other herbicideshave predominately preemergence (metolachlor) or postemergence(glyphosate) activity. These properties of particular herbicides areknown in the art and are readily determined by one of skill in the art.Further, one of skill in the art would readily be able to selectappropriate herbicides and application times for use with the transgenicplants of the invention and/or on areas in which transgenic plants ofthe invention are to be planted. “Preplant incorporation” involves theincorporation of compounds into the soil prior to planting.

Thus, the invention provides improved methods of growing a crop and/orcontrolling weeds such as, for example, “pre-planting burn down,”wherein an area is treated with herbicides prior to planting the crop ofinterest in order to better control weeds. The invention also providesmethods of growing a crop and/or controlling weeds which are “no-till”or “low-till” (also referred to as “reduced tillage”). In such methods,the soil is not cultivated or is cultivated less frequently during thegrowing cycle in comparison to traditional methods; these methods cansave costs that would otherwise be incurred due to additionalcultivation, including labor and fuel costs.

The methods of the invention encompass the use of simultaneous and/orsequential applications of multiple classes of herbicides. In someembodiments, the methods of the invention involve treating a plant ofthe invention and/or an area of interest (e.g., a field or area ofcultivation) and/or weed with just one herbicide or other chemical suchas, for example, a sulfonylurea herbicide.

The time at which a herbicide is applied to an area of interest (and anyplants therein) may be important in optimizing weed control. The time atwhich a herbicide is applied may be determined with reference to thesize of plants and/or the stage of growth and/or development of plantsin the area of interest, e.g., crop plants or weeds growing in the area.The stages of growth and/or development of plants are known in the art.For example, soybean plants normally progress through vegetative growthstages known as V_(E) (emergence), V_(C) (cotyledon), V₁ (unifoliate),and V₂ to V_(N). Soybeans then switch to the reproductive growth phasein response to photoperiod cues; reproductive stages include R₁(beginning bloom), R₂ (full bloom), R₃ (beginning pod), R₄ (full pod),R₅ (beginning seed), R₆ (full seed), R₇ (beginning maturity), and R₈(full maturity). Corn plants normally progress through the followingvegetative stages VE (emergence); V1 (first leaf); V2 (second leaf); V3(third leaf); V(n) (Nth/leaf); and VT (tasseling). Progression of maizethrough the reproductive phase is as follows: R1 (silking); R2(blistering); R3 (milk); R4 (dough); R5 (dent); and R6 (physiologicalmaturity). Cotton plants normally progress through V_(E) (emergence),V_(C) (cotyledon), V₁ (first true leaf), and V₂ to V_(N). Then,reproductive stages beginning around V₁₄ include R₁ (beginning bloom),R₂ (full bloom), R₃ (beginning boll), R₄ (cutout, boll development), R₅(beginning maturity, first opened boll), R₆ (maturity, 50% opened boll),and R₇ (full maturity, 80-90% open bolls). Thus, for example, the timeat which a herbicide or other chemical is applied to an area of interestin which plants are growing may be the time at which some or all of theplants in a particular area have reached at least a particular sizeand/or stage of growth and/or development, or the time at which some orall of the plants in a particular area have not yet reached a particularsize and/or stage of growth and/or development.

In some embodiments, the glyphosate/ALS inhibitor-tolerant plants of theinvention show improved tolerance to postemergence herbicide treatments.For example, plants of the invention may be tolerant to higher doses ofherbicide, tolerant to a broader range of herbicides (i.e., tolerance tomore ALS inhibitor chemistries), and/or may be tolerant to doses ofherbicide applied at earlier or later times of development in comparisonto an appropriate control plant. For example, in some embodiments, theglyphosate/ALS inhibitor-tolerant plants of the invention show anincreased resistance to morphological defects that are known to resultfrom treatment at particular stages of development. Thus, for example, aphenomenon known as “ear pinch” often results when maize plants aretreated with herbicide at a stage later than V5, V6, V7, V8, V9, V10,V11, V12, V13, or a later stage, whereas the glyphosate/ALSinhibitor-tolerant plants of the invention show a decreased incidence of“ear pinch” when treated at the same stage. Thus, the glyphosate/ALSinhibitor-tolerant plants of the invention find use in methods involvingherbicide treatments at later stages of development than were previouslyfeasible. Thus, plants of the invention may be treated with a particularherbicide that causes morphological defects in a control plant treatedat the same stage of development, but the glyphosate/ALSinhibitor-tolerant plants of the invention will not be significantlydamaged by the same treatment.

Different chemicals such as herbicides have different “residual”effects, i.e., different amounts of time for which treatment with thechemical or herbicide continues to have an effect on plants growing inthe treated area. Such effects may be desirable or undesirable,depending on the desired future purpose of the treated area (e.g., fieldor area of cultivation). Thus, a crop rotation scheme may be chosenbased on residual effects from treatments that will be used for eachcrop and their effect on the crop that will subsequently be grown in thesame area. One of skill in the art is familiar with techniques that canbe used to evaluate the residual effect of a herbicide; for example,generally, glyphosate has very little or no soil residual activity,while herbicides that act to inhibit ALS vary in their residual activitylevels. Residual activities for various herbicides are known in the art,and are also known to vary with various environmental factors such as,for example, soil moisture levels, temperature, pH, and soil composition(texture and organic matter). The glyphosate/ALS inhibitor-tolerantplants of the invention find particular use in methods of growing a cropwhere improved tolerance to residual activity of a herbicide isbeneficial.

For example, in one embodiment, the glyphosate/ALS inhibitor-tolerantplants of the invention have an improved tolerance to glyphosate as wellas to ALS inhibitor chemistries (such as sulfonylurea herbicides) whenapplied individually, and further provide improved tolerance tocombinations of herbicides such as glyphosate and/or ALS inhibitorchemistries. Moreover, the transgenic plants of the invention provideimproved tolerance to treatment with additional chemicals commonly usedon crops in conjunction with herbicide treatments, such as safeners,adjuvants such as ammonium sulfonate and crop oil concentrate, and thelike.

e. Safeners and Adjuvants

The term “safener” refers to a substance that when added to a herbicideformulation eliminates or reduces the phytotoxic effects of theherbicide to certain crops. One of ordinary skill in the art wouldappreciate that the choice of safener depends, in part, on the cropplant of interest and the particular herbicide or combination ofherbicides included in the synergistic herbicide composition. Exemplarysafeners suitable for use with the presently disclosed herbicidecompositions include, but are not limited to, those disclosed in U.S.Pat. Nos. 4,808,208; 5,502,025; 6,124,240 and U.S. Patent ApplicationPublication Nos. 2006/0148647; 2006/0030485; 2005/0233904; 2005/0049145;2004/0224849; 2004/0224848; 2004/0224844; 2004/0157737; 2004/0018940;2003/0171220; 2003/0130120; 2003/0078167, the disclosures of which areincorporated herein by reference in their entirety. The methods of theinvention can involve the use of herbicides in combination withherbicide safeners such as benoxacor, BCS(1-bromo-4-[(chloromethyl)sulfonyl]benzene), cloquintocet-mexyl,cyometrinil, dichlormid, 2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG191), fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole,isoxadifen-ethyl, mefenpyr-diethyl, methoxyphenone((4-methoxy-3-methylphenyl)(3-methylphenyl)-methanone), naphthalicanhydride (1,8-naphthalic anhydride) and oxabetrinil to increase cropsafety. Antidotally effective amounts of the herbicide safeners can beapplied at the same time as the compounds of this invention, or appliedas seed treatments. Therefore an aspect of the present invention relatesto the use of a mixture comprising glyphosate, at least one otherherbicide, and an antidotally effective amount of a herbicide safener.

Seed treatment is particularly useful for selective weed control,because it physically restricts antidoting to the crop plants. Thereforea particularly useful embodiment of the present invention is a methodfor selectively controlling the growth of weeds in a field comprisingtreating the seed from which the crop is grown with an antidotallyeffective amount of safener and treating the field with an effectiveamount of herbicide to control weeds. Antidotally effective amounts ofsafeners can be easily determined by one skilled in the art throughsimple experimentation. An antidotally effective amount of a safener ispresent where a desired plant is treated with the safener so that theeffect of a herbicide on the plant is decreased in comparison to theeffect of the herbicide on a plant that was not treated with thesafener; generally, an antidotally effective amount of safener preventsdamage or severe damage to the plant treated with the safener. One ofskill in the art is capable of determining whether the use of a safeneris appropriate and determining the dose at which a safener should beadministered to a crop.

In specific embodiments, the herbicide or herbicide combination appliedto the plant of the invention acts as a safener. In this embodiment, afirst herbicide or a herbicide mixture is applied at an antidotallyeffect amount to the plant. Accordingly, a method for controlling weedsin an area of cultivation is provided. The method comprises planting thearea with crop seeds or plants which comprise a first polynucleotideencoding a polypeptide that can confer tolerance to glyphosate operablylinked to a promoter active in a plant; and, a second polynucleotideencoding an ALS inhibitor-tolerant polypeptide operably linked to apromoter active in a plant. A combination of herbicides comprising atleast an effective amount of a first and a second herbicide is appliedto the crop, crop part, weed or area of cultivation thereof. Theeffective amount of the herbicide combination controls weeds; and, theeffective amount of the first herbicide is not tolerated by the cropwhen applied alone when compared to a control crop that has not beenexposed to the first herbicide; and, the effective amount of the secondherbicide is sufficient to produce a safening effect, wherein thesafening effect provides an increase in crop tolerance upon theapplication of the first and the second herbicide when compared to thecrop tolerance when the first herbicide is applied alone.

In specific embodiments, the combination of safening herbicidescomprises a first ALS inhibitor and a second ALS inhibitor. In otherembodiments, the safening effect is achieved by applying an effectiveamount of a combination of glyphosate and at least one ALS inhibitorchemistry. In still other embodiments, a safening affect is achievedwhen the glyphosate/ALS inhibitor-tolerant crops, crop part, crop seed,weed, or area of cultivation is treated with at least one herbicide fromthe sulfonylurea family of chemistries in combination with at least oneherbicide from the ALS family of chemistries (such as, for example, animidazolinone).

Such mixtures provides increased crop tolerance (i.e., a decrease inherbicidal injury). This method allows for increased application ratesof the chemistries post or pre-treatment. Such methods find use forincreased control of unwanted or undesired vegetation. In still otherembodiments, a safening affect is achieved when the glyphosate/ALSinhibitor-tolerant crops, crop part, crop seed, weed, or area ofcultivation is treated with at least one herbicide from the sulfonylureafamily of chemistry in combination with at least one herbicide from theimidazolinone family. This method provides increased crop tolerance(i.e., a decrease in herbicidal injury). In specific embodiments, thesulfonylurea comprises rimsulfuron and the imidazolinone comprisesimazethapyr. In other embodiments, glyphosate is also applied to thecrop, crop part, or area of cultivation.

As used herein, an “adjuvant” is any material added to a spray solutionor formulation to modify the action of an agricultural chemical or thephysical properties of the spray solution. See, for example, Green andFoy (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. Inderjit (Kluwer Academic Publishers,The Netherlands). Adjuvants can be categorized or subclassified asactivators, acidifiers, buffers, additives, adherents, antiflocculants,antifoamers, defoamers, antifreezes, attractants, basic blends,chelating agents, cleaners, colorants or dyes, compatibility agents,cosolvents, couplers, crop oil concentrates, deposition agents,detergents, dispersants, drift control agents, emulsifiers, evaporationreducers, extenders, fertilizers, foam markers, formulants, inerts,humectants, methylated seed oils, high load COCs, polymers, modifiedvegetable oils, penetrators, repellants, petroleum oil concentrates,preservatives, rainfast agents, retention aids, solubilizers,surfactants, spreaders, stickers, spreader stickers, synergists,thickeners, translocation aids, uv protectants, vegetable oils, waterconditioners, and wetting agents.

f. Additional Agricultural Chemicals

In addition, methods of the invention can comprise the use of aherbicide or a mixture of herbicides, as well as, one or more otherinsecticides, fungicides, nematocides, bactericides, acaricides, growthregulators, chemosterilants, semiochemicals, repellents, attractants,pheromones, feeding stimulants or other biologically active compounds orentomopathogenic bacteria, virus, or fungi to form a multi-componentmixture giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants which can be used in methodsof the invention include: insecticides such as abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, cartap,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin,cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin,dimethoate, dinotefuran, diofenolan, emamectin, endosulfan,esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin,fenvalerate, fipronil, flonicamid, flubendiamide, flucythrinate,tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos,halofenozide, hexaflumuron, hydramethylnon, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaflumizone, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,metofluthrin, monocrotophos, methoxyfenozide, nitenpyram, nithiazine,novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, profluthrin, pymetrozine, pyrafluprole, pyrethrin,pyridalyl, pyriprole, pyriproxyfen, rotenone, ryanodine, spinosad,spirodiclofen, spiromesifen (BSN 2060), spirotetramat, sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,triazamate, trichlorfon and triflumuron; fungicides such as fungicidessuch as acibenzolar, aldimorph, amisulbrom, azaconazole, azoxystrobin,benalaxyl, benomyl, benthiavalicarb, benthiavalicarb-isopropyl,binomial, biphenyl, bitertanol, blasticidin-S, Bordeaux mixture(Tribasic copper sulfate), boscalid/nicobifen, bromuconazole,bupirimate, buthiobate, carboxin, carpropamid, captafol, captan,carbendazim, chloroneb, chlorothalonil, chlozolinate, clotrimazole,copper oxychloride, copper salts such as copper sulfate and copperhydroxide, cyazofamid, cyflunamid, cymoxanil, cyproconazole, cyprodinil,dichlofluanid, diclocymet, diclomezine, dicloran, diethofencarb,difenoconazole, dimethomorph, dimoxystrobin, diniconazole,diniconazole-M, dinocap, discostrobin, dithianon, dodemorph, dodine,econazole, etaconazole, edifenphos, epoxiconazole, ethaboxam, ethirimol,ethridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole,fencaramid, fenfuram, fenhexamide, fenoxanil, fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferfurazoate,ferimzone, fluazinam, fludioxonil, flumetover, fluopicolide,fluoxastrobin, fluquinconazole, fluquinconazole, flusilazole,flusulfamide, flutolanil, flutriafol, folpet, fosetyl-aluminum,fuberidazole, furalaxyl, furametapyr, hexaconazole, hymexazole,guazatine, imazalil, imibenconazole, iminoctadine, iodicarb, ipconazole,iprobenfos, iprodione, iprovalicarb, isoconazole, isoprothiolane,kasugamycin, kresoxim-methyl, mancozeb, mandipropamid, maneb,mapanipyrin, mefenoxam, mepronil, metalaxyl, metconazole,methasulfocarb, metiram, metominostrobin/fenominostrobin, mepanipyrim,metrafenone, miconazole, myclobutanil, neo-asozin (ferricmethanearsonate), nuarimol, octhilinone, ofurace, orysastrobin,oxadixyl, oxolinic acid, oxpoconazole, oxycarboxin, paclobutrazol,penconazole, pencycuron, penthiopyrad, perfurazoate, phosphonic acid,phthalide, picobenzamid, picoxystrobin, polyoxin, probenazole,prochloraz, procymidone, propamocarb, propamocarb-hydrochloride,propiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin,pryazophos, pyrifenox, pyrimethanil, pyrifenox, pyrolnitrine,pyroquilon, quinconazole, quinoxyfen, quintozene, silthiofam,simeconazole, spiroxamine, streptomycin, sulfur, tebuconazole,techrazene, tecloftalam, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate, thiophanate-methyl, thiram, tiadinil,tolclofos-methyl, tolyfluanid, triadimefon, triadimenol, triarimol,triazoxide, tridemorph, trimoprhamide tricyclazole, trifloxystrobin,triforine, triticonazole, uniconazole, validamycin, vinclozolin, zineb,ziram, and zoxamide; nematocides such as aldicarb, oxamyl andfenamiphos; bactericides such as streptomycin; acaricides such asamitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol,dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin,fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; andbiological agents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. The weight ratios of these various mixing partners toother compositions (e.g., herbicides) used in the methods of theinvention typically are between 100:1 and 1:100, or between 30:1 and1:30, between 10:1 and 1:10, or between 4:1 and 1:4.

The present invention also pertains to a composition comprising abiologically effective amount of a herbicide of interest or a mixture ofherbicides, and an effective amount of at least one additionalbiologically active compound or agent and can further comprise at leastone of a surfactant, a solid diluent or a liquid diluent. Examples ofsuch biologically active compounds or agents are: insecticides such asabamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin,azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin,carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos,chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin,beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin,cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron,dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole,fenothicarb, fenoxycarb, fenpropathrin, fenvalerate, fipronil,flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701),flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid,indoxacarb, isofenphos, lufenuron, malathion, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron(XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate,phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine,pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060),sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos,tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb,thiosultap-sodium, tralomethrin, trichlorfon and triflumuron; fungicidessuch as acibenzolar, azoxystrobin, benomyl, blasticidin-S, Bordeauxmixture (tribasic copper sulfate), bromuconazole, carpropamid, captafol,captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride,copper salts, cyflufenamid, cymoxanil, cyproconazole, cyprodinil,(S)-3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide(RH 7281), diclocymet (S-2900), diclomezine, dicloran, difenoconazole,(S)-3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenyl-amino)-4H-imidazol-4-one(RP 407213), dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid (SZX0722), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil,flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin(HEC 5725), fluquinconazole, flusilazole, flutolanil, flutriafol,folpet, fosetyl-aluminum, furalaxyl, furametapyr (S-82658),hexaconazole, ipconazole, iprobenfos, iprodione, isoprothiolane,kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil,metalaxyl, metconazole, metomino-strobin/fenominostrobin (SSF-126),metrafenone (AC375839), myclobutanil, neo-asozin (ferricmethane-arsonate), nicobifen (BAS 510), orysastrobin, oxadixyl,penconazole, pencycuron, probenazole, prochloraz, propamocarb,propiconazole, proquinazid (DPX-KQ926), prothioconazole (JAU 6476),pyrifenox, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen,spiroxamine, sulfur, tebuconazole, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, triadimefon,triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycinand vinclozolin; nematocides such as aldicarb, oxamyl and fenamiphos;bactericides such as streptomycin; acaricides such as amitraz,chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad; and biologicalagents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. Methods of the invention may also comprise the use ofplants genetically transformed to express proteins toxic to invertebratepests (such as Bacillus thuringiensis delta-endotoxins). In suchembodiments, the effect of exogenously applied invertebrate pest controlcompounds may be synergistic with the expressed toxin proteins.

General references for these agricultural protectants include ThePesticide Manual, 13th Edition, C. D. S. Tomlin, Ed., British CropProtection Council, Farnham, Surrey, U.K., 2003 and The BioPesticideManual, 2^(nd) Edition, L. G. Copping, Ed., British Crop ProtectionCouncil, Farnham, Surrey, U.K., 2001.

In certain instances, combinations with other invertebrate pest controlcompounds or agents having a similar spectrum of control but a differentmode of action will be particularly advantageous for resistancemanagement. Thus, compositions of the present invention can furthercomprise a biologically effective amount of at least one additionalinvertebrate pest control compound or agent having a similar spectrum ofcontrol but a different mode of action. Contacting a plant geneticallymodified to express a plant protection compound (e.g., protein) or thelocus of the plant with a biologically effective amount of a compound ofthis invention can also provide a broader spectrum of plant protectionand be advantageous for resistance management.

Thus, methods of the invention employ a herbicide or herbicidecombination and may further comprise the use of insecticides and/orfungicides, and/or other agricultural chemicals such as fertilizers. Theuse of such combined treatments of the invention can broaden thespectrum of activity against additional weed species and suppress theproliferation of any resistant biotypes.

Methods of the invention can further comprise the use of plant growthregulators such as aviglycine, N-(phenylmethyl)-1H-purin-6-amine,ethephon, epocholeone, gibberellic acid, gibberellin A₄ and A₇, harpinprotein, mepiquat chloride, prohexadione calcium, prohydrojasmon, sodiumnitrophenolate and trinexapac-methyl, and plant growth modifyingorganisms such as Bacillus cereus strain BP01.

VI. Use as Selectable Markers and Methods of Transformation

In some embodiments of the invention, a construct of the inventioncomprising a GAT polynucleotide or an ALS inhibitor-tolerant polypeptidefunctions as a selectable marker, e.g., in a plant, bacteria,actinomycete, yeast, algae or other fungi. For example, an organism thathas been transformed with a vector including a GAT polynucleotide can beselected based on its ability to grow in the presence of glyphosate.Alternatively, an organism that has been transformed with a vectorcomprising an ALS-inhibitor-tolerant polynucleotide can be selectedbased on its ability to grown in the presence of an ALS inhibitor. Insome embodiments of the invention, a construct of the inventioncomprising a GAT polynucleotide and another herbicide-tolerancepolynucleotide (i.e., an polynucleotide encoding an ALSinhibitor-tolerant polypeptide, a polynucleotide encoding an HRApolypeptide, etc.) functions as a selectable marker, e.g., in a plant,bacteria, actinomycete, yeast, algae or other fungi. For example, anorganism that has been transformed with a vector including a GATpolynucleotide and another herbicide-tolerance polynucleotide can beselected based on its ability to grow in the presence of glyphosate andthe appropriate other herbicide. As demonstrated in Example 10 and FIG.7, such methods of selection allow one to evaluate expression of anypolynucleotide of interest at, for example, early stages in thetransformation process in order to identify potential problems withexpression. While any polynucleotide of interest can be employed, inspecific embodiments, an insecticidal gene is used.

A construct of the invention comprising a GAT polynucleotide and/or apolynucleotide encoding an ALS inhibitor-tolerant polypeptide mayexhibit a very high transformation efficiency, such as an efficiency ofat least 20%, 30%, 40%, 50%, or 60% or higher. In this manner, improvedmethods of transformation are provided. Moreover, when a construct ofthe invention comprises a GAT polynucleotide and/or ALSinhibitor-tolerant polynucleotide, the transformants that are obtainedmay exhibit a very high frequency of tolerance to glyphosate or ALSinhibitor, so that, for example, at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% of the transformants are tolerant to glyphosate and/oran ALS inhibitor. As used herein “transformation efficacy” is defined asthe percentage of the T0 events that were resistant to a specificconcentration of selection agent, such as glyphosate and/or an ALSinhibitor chemistry. When a construct of the invention comprises a GATpolynucleotide and/or ALS inhibitor-tolerant polynucleotide operablylinked to an enhancer such as, for example, a 35S enhancer, thetransformants that are obtained may exhibit a very high frequency oftolerance to glyphosate and/or ALS inhibitor, so that for example, atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of thetransformants are tolerant to glyphosate or ALS inhibitor. In addition,when a construct of the invention comprises a GAT polynucleotide and/oran ALS inhibitor-tolerant polynucleotide, the frequency oftransformation events in which only a single copy of the construct isinserted into the genome may be as high as at least 35%, 40%, 50%, 60%,70%, 80%, 90%, or higher. When a construct of the invention comprises aGAT polynucleotide and/or ALS inhibitor-tolerant polynucleotide operablylinked to an enhancer such as, for example, a 35S enhancer, thefrequency of transformation events in which only a single copy of theconstruct is inserted into the genome may be as high as at least 35%,40%, 50%, 60%, 70%, 80%, 90%, or higher. In this manner, the inventionalso provides improved methods of transformation. It is recognized thatwhen an enhancer is employed in the construct (such as S³⁵ enhancer)multiple copies could be used, including 1, 2, 3, 4, 5, 6 or more. Insuch methods, the transformants may be selected using glyphosate and/oran ALS inhibitor, or they may be selected using another compound, suchas another herbicide for which the transformed construct contains atolerance trait.

VII. Kits

The invention further provides a kit comprising at least one nucleicacid construct which comprises a polynucleotide which encodes apolypeptide that can confer glyphosate tolerance and/or a polynucleotideencoding an ALS inhibitor-tolerant polypeptide for use in creating aglyphosate/ALS inhibitor plant of the invention. In specificembodiments, the kit can comprise a polynucleotide encoding GAT or thekit can comprise a polynucleotide encoding GAT and a polynucleotideencoding an ALS inhibitor-tolerant polynucleotide (i.e., HRA). In someaspects a construct of the invention will comprise a T-DNA sequence. Theconstruct can optionally include a regulatory sequence (e.g., apromoter) operably linked to the polynucleotide conferring glyphosateresistance, where the promoter is heterologous with respect to thepolynucleotide and effective to cause sufficient expression of theencoded polypeptide to enhance the glyphosate tolerance of a plant celltransformed with the nucleic acid construct.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXPERIMENTAL Example 1 GAT-HRA Maize Plants are Tolerant of VariousHerbicides and Agricultural Chemicals

For Agrobacterium-mediated transformation of maize with an expressioncassette containing a GAT polynucleotide (SEQ ID NO:4) and an HRApolynucleotide (SEQ ID NO:66) operably linked to a constitutivepromoter, the method of Zhao is employed (U.S. Pat. No. 5,981,840, andPCT patent publication WO98/32326; the contents of which are herebyincorporated by reference). Expression cassettes were made thatcomprised a GAT polynucleotide and an HRA polynucleotide. In someexpression cassettes, the GAT and HRA polynucleotides were operablylinked to at least one copy of a 35S enhancer [SEQ ID NO:72]. In someexpression cassettes, the GAT and HRA polynucleotides were operablylinked to two or three copies of the 35S enhancer of SEQ ID NO:1. Insome expression cassettes, the GAT polynucleotide was operably linked toa ubiquitin promoter and the HRA polynucleotide was operably linked tothe native maize acetolactate synthase (ALS) promoter.

Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the GAT and HRA sequence to at least one cell ofat least one of the immature embryos (step 1: the infection step). Inthis step the immature embryos are immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos areco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos are incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos are cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.

Evaluation of Herbicide Tolerance

GAT-HRA maize plants were evaluated for tolerance to glyphosate andother herbicides. One protocol used in this evaluation was as follows.At the V2 stage (Ritchie and Hanway (1982), “How a corn plant develops”Spec. Rep. 48. (Coop. Ext. Ser., Iowa State Univ., Ames, Iowa)), plantheights were measured and herbicides were applied by spray application.Ten to fourteen days after the spray application, the transgenic plantswere evaluated for injury symptoms and measured for plant height againin order to determine plant growth rates. In one series of tests,GAT-HRA maize and control plants were treated (postemergence) with oneof the following herbicides: tribenuron (as Express®, at applicationrates of 0 and 200 grams active ingredient per hectare (g ai/ha));chlorimuron (as Classic®, at 0 and 200 g ai/ha); imazapyr (as Arsenal®,at 0 and 200 g ai/ha); metsulfuron-methyl (as Ally®, at 0.5 oz ai/ac(36.9 ml ai/ha) or 35 g ai/ha). For each treatment, the GAT-HRA maizeshowed no significant damage from the treatment, while the control maizewas severely damaged or killed.

GAT-HRA maize plants were also treated with various other herbicides andcombinations of herbicides as well as other agricultural chemicals,including: glyphosate (as WeatherMax®, at an application rate of 64.4 ozai/ac (4.7 L ai/ha)); rimsulfuron (as Matrix®, at an application rate of1.9 oz ai/ac (140 ml ai/ha)); sulfometuron-methyl (as Oust®, at anapplication rate of 4.5 oz ai/ac (332 ml ai/ha)); Basis® (a combinationof rimsulfuron and thifensulfuron-methyl, at an application rate of 1.4oz ai/ac (103 ml ai/ha)); and chlorpyrifos (as Lorsban®, at anapplication rate of 14.4 oz ai/ac (1.06 L ai/ha)). Plants were thenevaluated at various times after treatment, such as 10 or 14 days aftertreatment. Tolerant plants were those which showed little or no damagefollowing treatment with a herbicide or herbicide combination. Thisevaluation identified GAT-HRA plants which were tolerant to multiplesulfonylurea and glyphosate chemistries. GAT-HRA plants did not exhibitany significant differences in growth or seed set (i.e., yield) incomparison to control plants. Leaf samples were taken from GAT-HRAplants and used in quantitative PCR analysis and other analyses todetermine the number and arrangement of copies of the GAT and HRApolynucleotides that had been integrated into the plant genome.

Other protocols for evaluation included treatment with imidazolinoneherbicides, such as the commercial herbicide Lightning® (a combinationof imazapyr and imazethapyr), which was applied to GAT-HRA maize at theV2 leaf stage at four times the field label rate (250 g ai/ha). Plantswere evaluated fourteen days after the herbicide application, and fourof the six transgenic events tested showed no symptoms of injury fromthe herbicide. Another protocol for evaluation included treatment withimidazolinone herbicides as well as sulfonylurea and glyphosateherbicides. In these tests, GAT-HRA maize was treated with variouscombinations of Lightning® (imazapyr and imazethapyr), Basis®(rimsulfuron and thifensulfuron-methyl) and WeatherMAX® (glyphosatepotassium salt). These herbicides were applied at the V2 stage asfollows: Lightning® at twice the field label rate (1.8 oz ai/ac (133 mlai/ha)), Basis® at twice the field label rate (0.5 oz ai/ac (36.9 mlai/ha)) and WeatherMax® at four times the field label rate (43 ozai/ac). Fourteen days after the treatment application, the plants wereevaluated. Plants containing eleven of the twelve transgenic eventsshowed no herbicide injury symptoms. “Field label rate” refers to theapplication rate specified on the product label. Where the applicationrate for a particular situation is a range of rates, as used herein, theterm “field label rate” indicates the upper end of that range. Productlabel information for pesticides is available from the U.S.Environmental Protection Agency and is updated online at the urloaspub.epa.gov/pestlabl/ppls.own; product label information is alsoavailable online at the url www.cdms.net.

Further tests confirmed that the multiple herbicide tolerance of theseGAT-HRA maize plants was stably inherited. T1 seed was harvested from 20transgenic T0 plants that showed excellent herbicide tolerance ingreenhouse evaluations. As is known in the art, the tolerance of plantsto herbicide can vary with the environment, and herbicide treatments ina greenhouse environment can have a greater impact on treated plantsthan herbicide treatments in the field. Accordingly, the T1 seed wasfurther evaluated under field conditions. T1 plants were sprayed at theV4 leaf stage with four different herbicide treatment combinationsincluding sulfonylurea and glyphosate herbicides. In some tests, atransgenic control line was used that was known to be tolerant ofglyphosate but susceptible to other herbicides. The tested T1 plantsalso showed excellent herbicide tolerance under field conditions,confirming the stable inheritance of the herbicide tolerance.

Evaluation of Tolerance to Range of Herbicides

GAT-HRA maize plants were then evaluated to determine whether they werealso tolerant to other herbicides. A test of both preemergent andpostemergent herbicide application was conducted. Specifically, sevencorn seeds of each line to be evaluated were planted 1 cm deep in 5.5inch square plastic containers of Tama silt loam soil. Treatmentsdescribed in Tables 3 and 4 were applied before any watering. A weekafter emergence, seedlings were thinned so that each container had twouniform plants. Corn seedlings were watered and fertilized for rapidgrowth and grown with a 16-h light photoperiod. When the natural lightintensity fell below 500 μE/m²/s, it was supplemented by metal halidelights with 160 μE/m²/s photosynthetically active radiation. Temperaturewas maintained at 28±2° C. during the day and 22±2° C. at night.Relative humidity generally ranged from 50 to 90%.

The studies of preemergence herbicide application used commercialherbicide formulations. Spray mixtures were prepared using deionizedwater at room temperature and were stirred for at least 15 minutes.Treatments were sprayed 1 to 2 h after preparation. All treatments wereapplied in a spray volume of 374 L/ha with a flat fan nozzle at 51 cmwith spray pressure set at 138 kPa with a high pH, basic blend adjuvantto ensure solubilization. Plants were visually evaluated for injury andfresh shoots were weighed 4 weeks after treatment (results shown inTable 1). Crop injury was also estimated visually on a scale rangingfrom 0% to 100%, where 0% signifies no injury and 100% signifies plantdeath. Results are shown in Table 3 and are expressed as the mean offour replications. TABLE 3 Preemergence Effect of 34 ALS Herbicides onFresh Weight of GAT-HRA and Parent Inbred Corn patent Hetero- elitestiff zygous stalk GAT- inbred HRA ALS Inhibitor Class HerbicideTreatment* Fresh Weight (g) Untreated 36.7 47.0 Imidazolinone 200 g/haImazamethabenz-methyl 36.2 48.5 200 g/ha Imazethapyr 24.4 50.0 200 g/haImazamox 0.8 47.5 200 g/ha Imazapyr 0.3 44.2 200 g/ha Imazaquin 1.5 45.4Pyrimidinylthiobenzoate 200 g/ha Pyriminobac methyl 30.4 39.9 200 g/haPyrithiobac Na⁺ 0.0 48.6 Sulfonylaminocarbonyltriazolinone 200 g/haFlucarbazone Na⁺ 0.0 48.8 200 g/ha Propoxycarbazone Na⁺ 0.6 46.7Sulfonylurea 200 g/ha Amidosulfuron 14.2 47.7 200 g/ha Azimsulfuron 0.049.1 200 g/ha Bensulfuron-methyl 28.4 39.9 200 g/ha Chlorimuron-ethyl9.3 39.4 200 g/ha Chlorsulfuron 0.0 48.4 200 g/ha Ethametsulfuron-methyl1.6 50.1 200 g/ha Ethoxysulfuron 28.1 40.0 200 g/haFlupyrsulfuron-methyl Na⁺ 23.6 45.8 200 g/ha Foramsulfuron 30.6 39.1 200g/ha Halosulfuron-methyl 28.9 40.1 200 g/ha Iodosulfuron-methyl Na⁺ 24.452.5 200 g/ha Metsulfuron-methyl 0.0 41.1 200 g/ha Nicosulfuron 33.650.3 200 g/ha Primisulfuron-methyl 19.5 37.9 200 g/ha Prosulfuron 20.943.3 200 g/ha Rimsulfuron 29.8 45.3 200 g/ha Sulfometuron-methyl 0.047.3 200 g/ha Sulfosulfuron 1.0 50.6 200 g/ha Thifensulfuron-methyl 15.236.0 200 g/ha Triasulfuron 14.1 47.7 200 g/ha Tribenuron-methyl 32.146.8 200 g/ha Trifloxysulfuron Na⁺ 0.0 50.8 200 g/haTriflusulfuron-methyl 26.3 53.5 Triazolopyrimidine 200 g/haChloransulam-methyl 5.5 44.9 200 g/ha Flumetsulam 24.6 39.1

TABLE 4 Preemergence Effect of 34 ALS Herbicides on Visual Injury toGAT-HRA and Parent Inbred Corn Patent Hetero- elite stiff zygous stalkGAT- inbred HRA ALS Class Herbicide Treatment* % Visual InujuryImidazolinone 200 g/ha Imazamethabenz-methyl 61 15 200 g/ha Imazethapyr69 20 200 g/ha Imazamox 99 13 200 g/ha Imazapyr 99 30 200 g/ha Imazaquin99 28 Pyrimidinylthiobenzoate 200 g/ha Pyriminobac-methyl 83 20 200 g/haPyrithiobac Na⁺ 100 0 Sulfonylaminocarbonyltriazolinone 200 g/haFlucarbazone Na⁺ 100 40 200 g/ha Propoxycarbazone Na⁺ 100 100Sulfonylurea 200 g/ha Amidosulfuron 97 25 200 g/ha Azimsulfuron 100 40200 g/ha Bensulfuron-methyl 23 10 200 g/ha Chlorimuron-ethyl 100 10 200g/ha Chlorsulfuron 100 33 200 g/ha Ethametsulfuron-methyl 100 88 200g/ha Ethoxysulfuron 63 25 200 g/ha Flupyrsulfuron-methyl Na⁺ 87 90 200g/ha Foramsulfuron 5 7 200 g/ha Halosulfuron-methyl 30 15 200 g/haIodosulfuron-methyl Na⁺ 92 5 200 g/ha Metsulfuron-methyl 100 0 200 g/haNicosulfuron 43 0 200 g/ha Primisulfuron-methyl 68 7 200 g/haProsulfuron 68 9 200 g/ha Rimsulfuron 74 0 200 g/ha Sulfometuron-methyl100 6 200 g/ha Sulfosulfuron 100 18 200 g/ha Thifensulfuron-methyl 88 3200 g/ha Triasulfuron 89 12 200 g/ha Tribenuron-methyl 66 25 200 g/haTrifloxysulfuron Na⁺ 100 0 200 g/ha Triflusulfuron-methyl 100 5Triazolopyrimidine 200 g/ha Chloransulam-methyl 3 3 200 g/ha Flumetsulam13 13

The studies of postemergence herbicide application also used commercialherbicide formulations. Specifically, four corn seeds of each line to beevaluated were planted 1 cm deep in 5.5 inch square plastic containersof synthetic growth medium. Very young transgenic seedlings werepretreated with glyphosate to eliminate segregants which were sensitiveto glyphosate. Plants injured by the glyphosate treatment were removedand containers were thinned to two uniform plants each. Extra containerswere planted so that only containers with two uniform and uninjuredplants were used in the experiment. Corn seedlings were watered andfertilized for rapid growth and grown with a 16-h light photoperiod.When the natural light intensity fell below 500 μE/m²/s, it wassupplemented by metal halide lights with 160 μE/m²/s photosyntheticallyactive radiation. Temperature was maintained at 28±2° C. during the dayand 22±2° C. at night. Relative humidity generally ranged from 50 to90%.

Postemergence studies used commercial herbicide formulations and wereapplied two weeks after planting. Spray mixtures of herbicides wereprepared using deionized water at room temperature and were stirred forat least 15 minutes. Treatments were sprayed 1 to 2 h after preparation.All ALS herbicide treatments were applied in a spray volume of 374 L/hawith a flat fan nozzle at 51 cm with spray pressure set at 138 kPa andincluded a high pH, basic blend adjuvant to ensure solubilization andfoliar penetration. Glyphosate and glufosinate herbicide preparationsincluded adjuvant systems in their commercial formulations to ensurehigh foliar activity. Fresh shoots were weighed 2 weeks after treatment(data shown in Table 5), and plants were visually evaluated for injuryon a scale from 0% to 100% on which 0% indicates no injury and 100%indicates plant death (data shown in Table 6). Results are expressed inTables 5 and 6 as the mean of four replications. TABLE 5 PostemergenceEffect of 34 ALS Herbicides on Fresh Weight of GAT-HRA and Parent InbredCorn Hetero- Parent elite zygous stiff stalk GAT- inbred HRA ALS ClassHerbicide Treatment* Fresh Weight (g) Untreated 76.4 98.8 Imidazolinone200 g/ha Imazamethabenz-methyl 66.9 95.0 200 g/ha Imazethapyr 6.4 93.4200 g/ha Imazamox 2.2 90.9 200 g/ha Imazapyr 2.3 97.8 200 g/ha Imazaquin4.4 113.3 Pyrimidinylthiobenzoate 200 g/ha Pyriminobac-methyl 7.6 95.2200 g/ha Pyrithiobac Na⁺ 2.8 87.9 Sulfonylaminocarbonyltriazolinone 200g/ha Flucarbazone Na⁺ 3.1 86.0 200 g/ha Propoxycarbazone Na⁺ 1.9 101.2Sulfonylurea 200 g/ha Amidosulfuron 32.0 107.4 200 g/ha Azimsulfuron 1.587.9 200 g/ha Bensulfuron-methyl 9.0 103.3 200 g/ha Chlorimuron-ethyl10.1 95.5 200 g/ha Chlorsulfuron 1.7 105.2 200 g/haEthametsulfuron-methyl 5.6 98.0 200 g/ha Ethoxysulfuron 24.3 79.3 200g/ha Flupyrsulfuron-methyl 5.2 95.5 Na⁺ 200 g/ha Foramsulfuron 62.5 80.6200 g/ha Halosulfuron-methyl 58.1 94.5 200 g/ha Iodosulfuron-methyl Na⁺3.5 95.0 200 g/ha Metsulfuron-methyl 1.9 75.5 200 g/ha Nicosulfuron 62.794.7 200 g/ha Primisulfuron-methyl 16.6 101.0 200 g/ha Prosulfuron 53.691.3 200 g/ha Rimsulfuron 12.6 99.2 200 g/ha Sulfometuron-methyl 2.0104.5 200 g/ha Sulfosulfuron 4.0 105.5 200 g/ha Thifensulfuron-methyl4.2 86.6 200 g/ha Triasulfuron 6.4 90.3 200 g/ha Tribenuron-methyl 1.890.8 200 g/ha Trifloxysulfuron Na⁺ 1.0 86.6 200 g/haTriflusulfuron-methyl 2.1 110.9 Triazolopyrimidine 200 g/haChloransulam-methyl 6.8 104.4 200 g/ha Flumetsulam 12.8 103.8 Glycine3472 g ae/ha Glyphosate 1.0 97.4 Phosphinic Acid 1870 g/ha Glufosinateammonium 2.4 112.3

TABLE 6 Postemergence Effect of 34 ALS Herbicides on GAT-HRA and ParentInbred Corn Hetero- Parent elite zygous stiff stalk GAT- inbred HRA ALSClass Herbicide Treatment* % Visual Injury Imidazolinone 200 g/haImazamethabenz-methyl 11 16 200 g/ha Imazethapyr 97 9 200 g/ha Imazamox100 4 200 g/ha Imazapyr 100 1 200 g/ha Imazaquin 100 2Pyrimidinylthiobenzoate 200 g/ha Pyriminobac-methyl 93 4 200 g/haPyrithiobac Na⁺ 100 9 Sulfonylaminocarbonyltriazolinone 200 g/haFlucarbazone Na⁺ 100 14 200 g/ha Propoxycarbazone Na⁺ 100 9 Sulfonylurea200 g/ha Amidosulfuron 71 8 200 g/ha Azimsulfuron 100 20 200 g/haBensulfuron-methyl 93 14 200 g/ha Chlorimuron-ethyl 93 15 200 g/haChlorsulfuron 100 13 200 g/ha Ethametsulfuron-methyl 96 6 200 g/haEthoxysulfuron 88 15 200 g/ha Flupyrsulfuron-methyl 95 4 Na⁺ 200 g/haForamsulfuron 28 8 200 g/ha Halosulfuron-methyl 14 8 200 g/haIodosulfuron-methyl Na⁺ 100 1 200 g/ha Metsulfuron-methyl 100 14 200g/ha Nicosulfuron 24 0 200 g/ha Primisulfuron-methyl 75 5 200 g/haProsulfuron 25 29 200 g/ha Rimsulfuron 91 10 200 g/haSulfometuron-methyl 100 2 200 g/ha Sulfosulfuron 97 3 200 g/haThifensulfuron-methyl 98 10 200 g/ha Triasulfuron 97 6 200 g/haTribenuron-methyl 100 8 200 g/ha Trifloxysulfuron Na⁺ 100 29 200 g/haTriflusulfuron-methyl 100 8 Triazolopyrimidine 200 g/haChloransulam-methyl 96 3 200 g/ha Flumetsulam 85 3 Glycine 3472 g ae/haGlyphosate 100 10 Phosphinic Acid 1870 g/ha Glufosinate ammonium 99 1Additional Tests Including Other Agricultural Chemicals

Other protocols were used to evaluate whether GAT-HRA plants, inaddition to being more tolerant to various herbicides than controlplants, were also more tolerant to other agricultural chemicals thancontrol plants. For example, one protocol included treatment of GAT-HRAmaize with the sulfonylurea herbicides rimsulfuron andthifensulfuron-methyl in addition to treatment with the organophosphateinsecticide chlorpyrifos (Lorsban®). In this test, plants were evaluated14 days after treatment, and all 20 GAT-HRA transgenic plants showedgood to excellent tolerance to these chemicals while the control plantsshowed significant damage as a result of the treatments (see Table 8).Herbicide injury scores (also called tolerance scores) were assigned ona plot basis on a 1 to 9 scale with a rating of 9 indicating that plantsexhibited no injury symptoms and a rating of 1 indicating complete plantdeath. A rating of 5 indicates moderate leaf injury. The scale used hereis further explained in Table 7 below. TABLE 7 Herbicide injury scale (1to 9 scale scoring system) for maize Main Rating categories Detaileddescription 9 No Effect No crop reduction or injury 8 Slight Slight cropdiscoloration or stunting 7 Effect Some crop discoloration, stunting, orstunt loss 6 Crop injury more pronounced, but not lasting 5 ModerateModerate injury, crop usually recovers 4 Effect Crop injury morelasting, recovery doubtful 3 Lasting crop injury, no recovery 2 SevereHeavy crop injury and stand loss 1 Effect Crop nearly destroyed - A fewsurviving plants

TABLE 8 GAT HRA Transgenic Maize Plants Show Excellent Tolerance to anHerbicide as Measured by Tolerance Scores V4 Tolerance Scores Basis ®(rimsulfuron and thifensulfuron- Basis ® methyl), 1.0 oz (rimsulfuronand ai/ac (73.9 ml thifensulfuron- ai/ha); Lorsban ® methyl), 1.0 oz(chlorpyrifos), ai/ac (73.9 ml 14.4 oz ai/ac ai/ha); (1.06 L ai/ha);WeatherMax ® WeatherMax ® WeatherMax ® WeatherMax ® (glyphosate) 10.7 oz(glyphosate) 10.7 oz (glyphosate) 10.7 oz (glyphosate) 42.9 ozExpression ai/ac (790 ml ai/ac (790 ml ai/ac (790 ml ai/ac (3.17 LTransgenic Event cassette ai/ha) ai/ha) ai/ha) ai/ha) 2 4 9 9 9 7 4 3 99 9 9 3 3 9 9 9 5 10  3 8 9 9 7 Glyphosate 5.5 2.5 9 8.5 tolerantnon-GAT HRA Control

Fourteen days after the spray application, the 20 transgenic plants werealso measured for plant height on a plot basis. Plant heights of thesame four most tolerant GAT HRA events along with the non-GAT HRAcontrol were collected. The four transgenic events showed uniform plantgrowth on all the herbicide treatments. The non-GAT HRA control showed areduction in plant growth with the sulfonylurea treatments. The averageplot height results from these measurements are shown in Table 9. TABLE9 GAT HRA Transgenic Maize Plants Show Excellent Tolerance to anInsecticide as Measured by Average Plant Height V4 Average Plant Heights(inches) Basis ® (rimsulfuron and thifensulfuron- Basis ® methyl), 1.0oz (rimsulfuron and ai/ac (73.9 ml thifensulfuron- ai/ha); Lorsban ®methyl), 1.0 oz (chlorpyrifos), ai/ac (73.9 ml 14.4 oz ai/ac ai/ha);(1.06 L ai/ha); WeatherMax ® WeatherMax ® WeatherMax ® WeatherMax ®(glyphosate) 10.7 oz (glyphosate) 10.7 oz (glyphosate) 10.7 oz(glyphosate) 42.9 oz Transgenic Expression ai/ac (790 ml ai/ac (790 mlai/ac (790 ml ai/ac (3.17 L Event cassette ai/ha) ai/ha) ai/ha) ai/ha) 24 16 14 14 13 4 3 15 14 15 14 3 3 15 14 14 14 10  3 15 15 15 14Glyphosate 10 7.5 14.5 14 tolerant non-GAT HRA ControlComparison of Response of GAT-HRA Plants to a Range of Herbicide Doses

GAT-HRA maize plants were produced using various expression cassettesand assayed as described above for tolerance to multiple sulfonylureachemistries in combination with glyphosate (see Table 10). TABLE 10 GATHRA Maize Produced Using Various Expression Cassettes Is Tolerant toMultiple Sulfonylurea Chemistries Average Average number Average growthof inserts Percentage growth of non- integrated Total Number of highlysprayed sprayed in highly number of events Spray Expression tolerantplants plants tolerant of events with no Treatment cassette events(inches) (inches) events evaluated injury Matrix ® 3 25% 5.2 5.1 2.0 5614 (rimsulfuron) 1.9 oz ai/ac (140 ml ai/ha), WeatherMax ® (glyphosate)64.4 oz ai/ac (4.7 L ai/ha) Matrix ® 4 31% 5.2 5.1 1.6 51 16(rimsulfuron) 1.9 oz ai/ac (140 ml ai/ha), WeatherMax ® (glyphosate)64.4 oz ai/ac (4.7 L ai/ha) Matrix ® 5 27% 4.8 5.3 1.7 63 17(rimsulfuron) 1.9 oz ai/ac (140 ml ai/ha), WeatherMax ® (glyphosate)64.4 oz ai/ac (4.7 L ai/ha) Matrix ® 6 41% 4.5 5.0 1.4 182 75(rimsulfuron) 1.9 oz ai/ac (140 ml ai/ha), WeatherMax ® (glyphosate)64.4 oz ai/ac (4.7 L ai/ha) Basis ® 9 59% 2.6 3.1 1.4 27 16 (rimsulfuronand thifensulfuron- methly) 1.4 oz ai/ac (103 ml ai/ha), Lorsban ®(chlorpyrifos) 14.4 oz ai/ac (1.06 L ai/ha), WeatherMax ® (glyphosate)64.4 oz ai/ac (4.7 L ai/ha) Oust ® 9 71% 4.2 4.8 1.1 146 104(sulfometuron- methly) 4.5 oz ai/ac (322 ml ai/ac), WeatherMax ®(glyphosate) 64.4 oz ai/ac (4.7 L ai/ha)

Example GAT-HRA Soybean Plants are Tolerant of Various Herbicides andAgricultural Chemicals

Transformation and Regeneration of Transgenic Plants

Soybean embryos were bombarded with an expression cassette containingGAT (SEQ ID NO:68) and HRA polynucleotides (SEQ ID NO:65) operablylinked to a constitutive promoter, as follows. The promoter used for theGAT sequence is the SCP1 promoter, and the promoter used for the HRAsequence is the SAMS promoter. The 2 promoter/gene combinations arearranged in a tandem orientation with the HRA promoter/gene downstreamof the GAT promoter/gene. To induce somatic embryos, cotyledons lessthan 4 mm in length dissected from surface-sterilized, immature seeds ofthe soybean variety Jack were cultured in the light or dark at 26° C. onan appropriate agar medium for six to ten weeks. Somatic embryosproducing secondary embryos were then excised and placed into a suitableliquid medium. After repeated selection for clusters of somatic embryosthat multiplied as early, globular-staged embryos, the suspensions weremaintained as described below. Here, the herbicide-tolerance traits thatshould be possessed by the transformed plants were used as selectablemarkers.

Soybean embryogenic suspension cultures were maintained in 35 ml liquidmedia on a rotary shaker, 150 rpm, at 26° C. with fluorescent lights ona 16:8 hour day/night schedule. Cultures were subcultured every twoweeks by inoculating approximately 35 mg of tissue into 35 ml of liquidmedium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327: 70-73, U.S. Pat. No. 4,945,050).

To 50 μl of a 60 mg/ml 1 μm gold particle suspension was added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation was then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles were then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensionwere sonicated three times for one second each. Five microliters of theDNA-coated gold particles were then loaded on each macro carrier disk.

Approximately 150-200 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue were normally bombarded. Membrane rupture pressurewas set at 1100 psi (77.356 kg/cm), and the chamber was evacuated to avacuum of 28 inches mercury. The tissue was placed approximately 8.89 cmaway from the retaining screen and bombarded three times. Followingbombardment, the tissue was divided in half and placed back into liquidand cultured as described above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media, and eleven to twelve days post-bombardment with fresh mediacontaining 30-50 mg/L hygromycin and 100 ng/ml chlorsulfuron was used asa selection agent. This selective media was refreshed weekly. Seven toeight weeks post-bombardment, green, transformed tissue was observedgrowing from untransformed, necrotic embryogenic clusters. Isolatedgreen tissue was removed and inoculated into individual flasks togenerate new, clonally propagated, transformed embryogenic suspensioncultures. Each new culture derived from a separate area of transformedtissue was treated as an independent transformation event; theindividual culture as well as the initial (T0) plant(s) derived from asingle area of transformed tissue as well as its descendants weregenerally to represent a single “event.” These suspensions were thensubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Herbicide Treatments

Young regenerated transgenic plants were sprayed with 1× glyphosate (1×rate of glyphosate is 1120 g/ha of glyphosate isopropylamine) to cullsegregants. Four replications were performed for each treatment. Plantswere then treated with various herbicides, including glyphosate andALS-inhibitor herbicides. When treated with tribenuron-methyl herbicide,the GAT- and HRA-containing transgenic soybeans treated at 0 and 35 g/hashowed no significant damage, while herbicide-treated non-transgeniccontrol soybeans were killed by the treatment. Plants were then treatedwith glyphosate at 8× and tribenuron-methyl at 35 g/ha as well asrimsulfuron at 35 g/ha, and similar results were obtained.

Six soybean seeds from each variety to be assayed were planted 1 cm deepin 5.5 (13.97 cm) inch square plastic container of a synthetic growthmedium. Very young transgenic seedlings were pretreated with glyphosateto eliminate segregants that were not tolerant to glyphosate; plantsinjured by this treatment were removed. When possible, containers werethinned to two uniform plants. Non-transgenic lines were thinned to twouniform plants per container. Soybean seedlings were watered andfertilized for rapid growth. The plants were grown with a 16-h lightphotoperiod, and when natural light intensity fell below 500 μE/m²/s itwas supplemented with metal halide lights with 160 μE/m²/sphotosynthetically active radiation. Temperature was maintained at 28±2°C. during the day and 22±2° C. at night. Relative humidity generallyranged from 50 to 90%.

These postemergence studies used commercial herbicide formulations andwere applied approximately two weeks after planting. Spray mixtures weremade using deionized water at room temperature and were stirred for atleast 15 minutes. Treatments were sprayed 1 to 2 h after preparation.The 35 g/ha rimsulfuron and tribenuron-methyl herbicide treatments wereapplied in a spray volume of 374 L/ha with a flat fan nozzle at 51 cmwith spray pressure set at 138 kPa and included a high pH, basic blendadjuvant to ensure solubilization and foliar penetration. The commercialformulation of glyphosate treatment was also applied in a spray volumeof 374 L/ha with a flat fan nozzle at 51 cm with spray pressure set at138 kPa. Plants were visually evaluated for injury on a scale of 0%being no injury and 100% being plant death. Results are expressed belowin Table 11 as the mean of four replications. TABLE 11 PostemergenceEffect of Glyphosate and Two Sulfonylurea Herbicides on GAT-HRA SoybeansTribenuron- Line # Glyphosate methyl Rimsulfuron (and (8960 g/ha) (35g/ha) (35 g/ha) Event # mean) % Visual Injury 64 Mean 14 43 73 47 10 3473 41 20 55 73 60 Mean 15 55 83 41 13 38 80 70 9 40 80 35 9 49 85 11 1171 83 22 11 45 76 75 14 69 86 79 18 69 86 07 19 46 84 77 19 56 90 46 2049 78 82 20 61 90 84 22 71 83 61 Mean 20 54 82 99 10 34 79 47 15 36 7862 6 43 71 49 9 54 84 02 10 41 80 14 10 65 90 18 10 69 89 51 10 60 83  02B 10 40 70 05 11 55 78 60 16 45 73 34 23 76 90 24 60 63 90 33 84 8095 59 Mean 38 59 87 28 37 60 86 21 40 58 88 STS ® Mean 100 87 97 25 10087 97 46 100 88 97 Wild Type Mean 100 97 97 82 100 97 97 Jack 100 97 97

Example 3 Transgenic GAT-HRA Cotton is Tolerant of Several Herbicides

Cotton (Gossypium hirsutum) Coker 312 was transformed with a GATpolynucleotide (gat 4621) together with an Hra polynucleotide (SEQ IDNO:86) both operably linked to strong, constitutive plant viralpromoters. The promoter linked to gat4621 contains a duplicated portionof the Strawberry Vein Banding Virus transcript promoter (Wang et al.Virus Genes 20: 11-17, 2000; Genbank X97304). The Hra gene was driven bya duplicated portion of the Mirabilis Mosaic Caulimovirus full-lengthtranscript promoter (U.S. Pat. No. 6,420,547; Dey and Maiti, Transgenics3:61-70, 1999). The transformation procedure used Agrobacteriumtumefaciens containing an expression cassette with the genes to betransferred and cotyledon explant derived callus with some capacity toundergo embryogenesis. The callus was exposed to Agrobacterium for 48-72hours. The callus was then exposed to selection for transformed cells on50-200 μg/l chlorosulfuron and/or 50-450 μM glyphosate in solid medium.Glyphosate can also be used in a concentration range of about 5 to about450 μM. Selection was applied until somatic embryos formed, andselection was again applied at the embryo germination step, andoptionally during rooting of plantlets. Over 450 GAT-HRA transformationevents were produced using selection with both chlorsulfuron andglyphosate.

Transformed cotton plants were rooted and then transferred to soil forfurther growth. Plants were then subjected to herbicide spray treatmentsin a greenhouse. In one treatment, glyphosate was sprayed over the topof plants at the 4-6 leaf stage of development at an application rate of1.5 lb acid equivalent per acre (2.58 Kg acid equivalent per hectare).Untransformed Coker 312 control plants were dead 2 weeks afterglyphosate application. In contrast, approximately 50% of the GAT-HRAtransgenic plants (each corresponding to a separate transformationevent) showed no deleterious symptoms from the glyphosate treatment 14days after application. Plants transformed with both GAT and HRA werefurther subjected to an “over the top” application of the sulfonylureaherbicide rimsulfuron at a rate of 16 g active ingredient/hectare.Again, approximately 35% of the transgenic plants showed no deleterioussymptoms from the dual herbicide application 14 days after therimsulfuron application and 28 days after the glyphosate application.Untransformed Coker 312 plants showed severe damage from the rimsulfuronapplication, even in the absence of any glyphosate application. Infurther experiments 32 g ai/ha rimsulfuron were applied to the cotton.

The presence of each of the GAT and HRA polynucleotides was furtherconfirmed in the herbicide-resistant transgenic plants using polymerasechain reaction (“PCR”) assays and using Western blot analysis to detectthe expressed GAT and HRA polypeptides.

Example 4 Formulation of Homogenous Granule Blends

A herbicidal composition useful for the present invention may beformulated in any suitable manner, for example, as a “homogeneousgranule blend” (see, e.g., U.S. Pat. No. 6,022,552, entitled “UniformMixtures of Pesticide Granules”). A herbicidal composition formulated asa homogeneous granule blend according to U.S. Pat. No. 6,022,552 can beprepared by shaking or otherwise mixing two or more groups ofsubstantially cylindrical granules typically made by extrusion orpelletization, wherein one group has an active ingredient contentcomprising at least one herbicide, and one or more other groups have adifferent active ingredient content or inert content, the granuleswithin each group having substantially uniform diameters andlongitudinal lengths of from 1 to 8 times the diameter with the averagelength of the granules being from 1.5 to 4 times the diameter, and theaverage diameter of each group differing from another group by no morethan 30%. In some embodiments, each granule group comprises a registeredformulation product containing a single active ingredient, which is forexample, a herbicide, fungicide, and/or an insecticide. “Substantiallycylindrical” is rod like or tubular wherein the cross-sectional shapemay be circular, octagonal, rectangular, or any other conceivable shapeand wherein the longitudinal surface is spiral, curved, or straight. Thedifference in average diameter is calculated by subtracting the averagediameter of the granules in the group having the smaller diameter fromthe average diameter of the granules in the group having the largerdiameter, then dividing the calculated difference by the averagediameter of the granules in the group having the smaller diameter, andfinally multiplying the calculated quotient by 100%.

The uniformity of a “homogenous granule blend” can be optimized bycontrolling the relative sizes and size distributions of the granulesused in the blend. Density differences are comparatively unimportant(see, e.g., Rhodes (1990) Principles of Powder Technology, pp. 71-76(John Wiley & Sons)). The diameter of extruded granules is controlled bythe size of the holes in the extruder die, and a centrifugal siftingprocess may be used to obtain a population of extruded granules with adesired length distribution (see, e.g., U.S. Pat. No. 6,270,025).Preferably the average diameter of each granule group differs fromanother group by no more than 20%, more preferably by no more than 10%.Also preferably the longitudinal length of each group is form 1.5 to 4times the diameter of the granules.

The active ingredient in each formulation has an associated tolerancefor variability based on guidelines of the Food and AgricultureOrganization of the United Nations (FAO), as shown below in Table 12.TABLE 12 FAO Nominal Concentration Guidelines for Active Ingredient in aFormulation Nominal Concentration FAO Range (as % of (= N) Nominal) N ≦2.5% ±25% 2.5% ≦ N ≦ 10% ±10% 10% ≦ N ≦ 25%  ±6% 25% ≦ N ≦ 50%  ±5% N >50% ±25 g/kg

The active ingredient content in a homogenous granule blend isdetermined based on the active ingredient content of the componentgranules and the ratio in which the component granules are mixed.Homogenous granule blends are manufactured assuming that the nominalvalues of active ingredients of the blend components are correct.Because of the real-life variability associated with assays of activeingredients as well as variability in mixing and sampling, procedureswere developed to calculate ranges for the active ingredient content ina homogenous granule blend, as follows.

-   -   1. Define the registered FAO specifications for each of the        blend components (“% AI in Component”).    -   2. Apply the FAO tolerance to establish manufacturing limits for        the amount of each component in the blend (“% Component in        Blend”).    -   3. Calculate the maximum limit for the active ingredient in the        blend (“% AI in the Blend”) by multiplying the maximum limit for        “% Al in Component” with the maximum limit for “% Component in        Blend.” The minimum and nominal calculations are similarly done.

Examples of the calculations for several homogenous granule blendproducts follow. The last column in each example table shows thestandard FAO assay range that would apply to a traditional premixproduct containing the same active ingredient content as the homogenousgranule blend in the example. The broader range for the homogenousgranule blend product (“% AI in Blend”) allows for the variabilityintroduced by using a registered product with an associated range ofactive ingredient content as a component in the product. TABLE 13Calculations for product “DPX-CDQ73 39.1WG” Homogenous Granule Blend* %Al in % Component in Compare to FAO Components Blend % Al in Blendtolerance for a (A) (B) (A × B) premix % metsulfuro- % Ally 20 PX in %metsulfuron- % metsulfuron- methyl in Ally DPX-CDQ73 methyl in DPX-methyl in 20PX Blend CDQ73 Blend traditional premix 21.2 max 66.7 max14.4 max 13.8 max 20.0 ± 6% 65.2 ± 25 g/kg 13.0 nominal 13.0 nominal18.8 min 62.7 min 11.8 min 12.3 min % tribenuron- % Quantum 75PX %tribenuron- % tribenuron- methyl in in DPX-CDQ73 methyl in DPX- methylin Quantum 75PX Blend CDQ73 Blend traditional premix 77.5 max 36.5 max28.3 max 27.4 max 75 ± 25 g/kg 34.8 ± 5% 26.1 nominal 26.1 nominal 72.5min 33.1 min 24.0 min 24.8 min*= A blend of 65.2% Ally 20PX and 34.8% Quantum 75PX. This blend is soldcommercially as BiPlay and DP911.

TABLE 14 Calculations for “DPX-CDQ74 51.5WG” Homogenous Granule Blend**% % Al in Component in Compare to FAO Components Blend % Al in Blendtolerance for a (A) (B) (A × B) premix % metsulfuron- % Ally 20 PX %metsulfuron- % metsulfuron- methyl in Ally in DPX- methyl in DPX- methylin 20PX CDQ73 CDQ73 Blend traditional premix Blend 21.2 max 44.9 max 9.5max 9.4 max 20.0 ± 6% 42.8 ± 5% 8.6 nominal 8.6 nominal 18.8 min 40.7min 7.7 min 7.7 min % thifensulfuron- % Harmony % thifensulfuron- %thifensulfuron- methyl in 75PX in DPX- methyl in DPX- methyl in Harmony75PX CDQ74 CDQ74 Blend traditional premix Blend 77.5 max 59.7 max 46.3max 45.0 max 75 ± 25 g/kg 57.2 ± 25 g/kg 42.9 nominal 42.9 nominal 72.5min 54.7 min 39.7 min 40.8 min**= A blend of 42.8% Ally 20PX and 57.2% Harmony 75PX. This blend issold commercially as Finish and DP928.

TABLE 15 “DPX-FKU22 60WG” Homogenous Granule Blend*** % Al in %Component in Compare to FAO Components Blend % Al in Blend tolerance fora (A) (B) (A × B) premix % % Lexus 50PX in % flupyrsulfuron %flupyrsulfuron flupyrsulfuron DPX-FKU22 methyl in DPX- methyl in methylin Blend FKU22 Blend traditional premix Lexus 50PX 52.5 max 62.5 max32.8 max 31.5 max 50.0 ± 5% 60.0 ± 25 g/kg 30.0 nominal 30.0 nominal47.5 min 57.5 min 27.3 min 28.5 min % tribenuron- % Quantum 75PX %tribenuron- % tribenuron- methyl in in DPX-FKU22 methyl in DPX- methylin Quantum 75PX Blend FKU22 Blend traditional premix 77.5 max 42.0 max32.6 max 31.5 max 75 ± 25 g/kg 40.0 ± 5% 30.0 nominal 30.0 nominal 72.5min 38.0 min 27.6 min 28.5 min***= A blend of 60% Lexus 50PX and 40% Quantum 75PX. This blend is soldcommercially as DP953.

Procedures were also developed to determine whether a particularhomogenous granule blend falls within the desired ranges. Homogenousgranule blends are random mixtures of granules; therefore, in order toaccurately represent the composition, a certain number of granules mustbe evaluated. The minimum number of granules for the sample can beestimated using a statistical equation (see Rhodes (1990), Principles ofPowder Technology (John Wiley & Sons), pp. 71-76),s ² =P(100−P)/nwhere s=standard deviation for the component proportion in the blend,P=weight percent of the component, and n=number of granules in thesample (˜400 1-mm diameter granules=1 gram). The sample size required torepresent the blend composition for a particular chosen level ofvariability can be obtained by solving for n and converting this valueto grams by dividing ‘n’ by the average number of 1-mm paste extrudedgranules in a 1-gram sample (e.g., 400).

If the standard deviation in this calculation is based on the FAOtolerance for the amount of a component in a particular granule blend, aminimum sample size for that granule blend can be calculated. For 95%confidence, the tolerance around the “% component in the blend” is setas 2 standard deviations. It is understood that this is a theoreticalstatistical estimate.

For a granule blend to be a feasible commercial product, the statisticalsample size must be equivalent to or smaller than the smallest amountthat would be measured by a farmer or applicator, typically a hectaredose. Examples of a sample size calculation for the granule blendsdiscussed above is shown below. TABLE 16 Minimum statistical sample sizecalculation for DPX-CDQ73 39.1WG Blend P Statistical Blend (% FOAtolerance for Minimum Component componen tin Blend) % component SampleSize Ally 20PX 65.2 ±25 g/kg 4 grams (±3.8% relative) Quantum 75PX 34.8±5% relative 8 gramsDetailed Calculation for Minimum Sample Size for DPX-CDQ73 39.1 WGBlend:

For the minor blend component (Quantum 75PX) the FAO tolerance of ±5%relative gives a range of: 5%×34.8=1.7

For 95% confidence, 2 standard deviations are set at=1.7 giving astandard deviation of s=0.85 for the calculation:s ² P(1−P)/n=(0.85)2=(65.2×34.8)/n

n=3140 granules=7.8 grams (based on 400 granules/gram)

The calculated minimum statistical sample size of about 8 grams is lessthan the product use rate of 38 g/ha.

A homogenous granule blend is considered to be “homogenous” when it canbe subsampled into appropriately sized aliquots and the composition ofeach aliquot will meet the required assay specifications. To demonstratehomogeneity, a large sample of the homogenous granule blend is preparedand is then subsampled into aliquots of greater than the minimumstatistical sample size. A second sample of the blend is prepared andsubjected to a simulated transportation test (ASTM D 4728-87, StandardMethod for Random Vibration Testing of Shipping Containers) and thensubsampled into aliquots. The aliquots are analyzed for activeingredient using liquid chromatography. The simulated transportationtest shakes the bottle at specific frequencies for a standard amount oftime, giving the granules an opportunity to move about. When the granuleblend components are not appropriately sized, segregation occurs and thealiquot compositions vary unacceptably.

Aliquot analysis data from the homogeneity tests for DPX-CDQ73 39.1 WGBlend are shown below in Table 17. All data points tested in the granuleblend aliquots fell within their respective calculated specificationranges, indicating that the blend was homogenous. TABLE 17 Analysis ofDPX-CDQ73 39.1WG Blend as made after simulated shipment. % % %metsulfuron- % tribenuron- metsulfuron- tribenuron- methyl methyl inmethyl in methyl in in DPX- DPX-CDQ73 Aliquot DPX- DPX- CDQ73 BlendBlend after (20 CDQ73 CDQ73 after simulated simulated grams) Blend Blendshipment shipment Proposed 11.8-14.4 24.0-28.3 11.8-14.4 24.0-28.3 assayrange 1 13.43 25.95 13.83 26.84 2 13.87 25.05 13.84 24.43 3 13.79 25.8813.28 27.2  4 13.36 26.8  13.67 26.25 5 13.49 27.37 13.57 27.33

Homogenous granule blends have been manufactured in a batch processusing a roll-type mixer. Once mixed, the granule blend is dispensed intoappropriate containers (bottles, bags, etc.) for commercial sale.Testing of the manufactured homogenous granule blend DPX-CDQ73 WG showedthat all data for the different batches of blend were within therespective proposed assay ranges for active ingredient.

Example 5 Evaluation of Levels of Glyphosate and ALS Inhibitor HerbicideEfficacy Among Soybean Plants Carrying Different GAT and HRA Sequences

Three GAT7 events in soybean were compared to four selected GAT11soybean events to determine if differences could be detected fortolerance to high rates of glyphosate+sulfonylurea treatments. Acrossthe treatment combinations, the GAT7 construct had significantly lessspray response compared to the GAT11 constructs at 7, 14, and 28 DAS.Within the 8× Touchdown® +1× Resolve®+2× Express® treatment, and the 8×Touchdown®+2× Resolve®+4× Express® treatment, GAT7 event EAFS 3560.4.3had the lowest spray response scores compared to all other events at 7,14, and 28 DAS.

Materials and Methods

For each treatment, three replications of two 12 foot plots of threeselected GAT7 events and four selected GAT11 events were grown in a RCBdesign (blocked by treatment) at two locations in Hawaii. Individuallines, events, and construct tested are listed below in Table 18. TABLE18 Entry Name GAT Event Construct Construct Description JH12862353 GAT11EAFS 3861.2.3 PHP22021A H2B:GAT4618::3(35Senh) + :SCP:HRA (DV)JH12862357 GAT11 EAFS 3862.2.5 PHP22021A H2B:GAT4618::3(35Senh) +:SCP:HRA (DV) JH12862359 GAT11 EAFS 3862.2.5 PHP22021AH2B:GAT4618::3(35Senh) + :SCP:HRA (DV) JH12862360 GAT11 EAFS 3862.2.5PHP22021A H2B:GAT4618::3(35Senh) + :SCP:HRA (DV) JH12862361 GAT11 EAFS3862.2.5 PHP22021A H2B:GAT4618::3(35Senh) + :SCP:HRA (DV) JH12862364GAT11 EAFS 3862.4.2 PHP22021A H2B:GAT4618::3(35Senh) + :SCP:HRA (DV)JH12862365 GAT11 EAFS 3862.4.2 PHP22021A H2B:GAT4618::3(35Senh) +:SCP:HRA (DV) JH12862405 GAT11 EAFS 3876.8.15 PHP22117AH2B:GAT4621::3(35Senh) + :SCP:HRA (DV) JH12862406 GAT11 EAFS 3876.8.15PHP22117A H2B:GAT4621::3(35Senh) + :SCP:HRA (DV) JH12862528 GAT7 EAFS3560.4.3 PHP20163A SCP:GAT4601::SAMS:HRA JH12862529 GAT7 EAFS 3559.2.1PHP20163A SCP:GAT4601::SAMS:HRA JH12862531 GAT7 EAFS 3561.1.1 PHP20163ASCP:GAT4601::SAMS:HRAThe three different treatments applied at the V3 growth stage were; 1.8×Touchdown® Hi-Tech (8630.55 g/ha a.i. glyphosate)+1× Resolve® (35.0 g/haa.i. rimsulfuron)+2× Express® (17.5 g/ha tribenuron), 2.8× Touchdown®Hi-Tech (8630.55 g/ha a.i. glyphosate)+2× Resolve® (70.0 g/ha a.i.rimsulfuron )+4× Express® (35.0 g/ha tribenuron), and 3. UnsprayedControl. All spray treatments also contained a 1× non-ionic surfactantand ammonium sulfate. At 7, 14, and 28 days after spraying, plots weregiven a visual spray damage rating based upon observed chlorosis,necrosis, and/or plant stunting (0%=no observed effect to 100%=entireplot deceased). Visual rating data were subject to ANOVA and meanseparation using SAS.Results and Discussion

Across the three treatments, the round of GAT (7 vs. 11), DNA construct,event, treatment, GAT*treatment, construct*treatment, andevent*treatment were significantly different at 7 DAS and 14 DAS (datanot shown). At 28 DAS, the GAT round, construct, event, treatment,GAT*treatment, and event*treatment effects were significantly different(data not shown). The GAT7 lines had significantly less response notedacross the three treatments at 7, 14, and 28 DAS (data not shown).

Within the 8× Touchdown®+1× Resolve®+2× Express® treatment, the GAT7construct PHP20163A had significantly lower spray response ratingscompared to GAT11 construct PHP22021A at 7, 14, and 28 DAS (data notshown). PHP20163A had significantly more tolerance to this treatmentcompared to PHP22117A only at 28 DAS (data not shown). Among the eventscompared, GAT7 event EAFS 3560.4.3 had the most initial toleranceobserved at 7 and 14 DAS, and had the best recovery score at 28 DAS(data not shown). In examining the differences between least squaresmeans (LSMeans), each of the three GAT7 events had significantly lessspray response at 7 DAS compared to GAT11 EAFS 3861.2.3 and GAT11 EAFS3862.2.5, and were rated statistically similar to GAT11 EAFS 3862.2.5and GAT11 EAFS 3876.8.1 (data not shown). At 14 DAS, the 3 GAT7 eventshad significantly less response scores compared to GAT11 EAFS 3862.4.2,but only GAT7 EAFS 3560.4.3 had significantly less response compared toEAFS 3861.2.3 and EAFS 3862.2.5 (data not shown). At 28 DAS, all threeGAT7 events had significantly better recovery compared to GAT11 eventsEAFS 3861.2.3, EAFS 3862.4.2, and EAFS 3876.8.1 (data not shown). Inaddition, GAT7 event EAFS 3560.4.3 had significantly less spray responsecompared to GAT11 event EAFS 3862.2.5 at 28 DAS (data not shown).

In examining the 8× Touchdown®+2× Resolve®+4× Express® treatment, GAT7PHP20163A had significantly less response compared to GAT11 PHP22021 at7, 14, and 28 DAS, and PHP20163A had significantly less responsecompared to GAT11 PHP22117A at 28 DAS (data not shown). Among theevents, GAT7 EAFS 3560.4.3 had significantly better tolerance comparedto all other events at 7 and 14 DAS, and GAT7 EAFS 3559.2.1 and EAFS3560.4.3 had significantly lower spray response compared to all otherevents at 28 DAS (data not shown). Comparing the differences in LSMeans,at 7 DAS and 14 DAS, GAT7 EAFS 3560.4.3 had significantly less responsecompared to all the GAT 11 events (data not shown). The other 2 GAT7events were significantly lower in response compared to GAT 11 eventEAFS 3862.4.2 at 7 and 14 DAS (data not shown). At 28 DAS, GAT7 eventsEAPS 3559.2.1 and EAFS 3560.4.3 had significantly better recoverycompared to all the GAT11 events (data not shown). GAT7 event EAFS3561.1.1 was only significantly better than GAT11 event EAFS 3862.4.2(data not shown).

Example 6 Robustness Trial Data Analysis in Soybean

The interactions between sulfonylurea and imidazolinone under fieldtrial conditions have been studied. Antagonism between the sulfonylureaand imidazolinone chemistries has been seen in the past on commercialSTS® soybean varieties. An SU like thifensulfuron on STS® soy isnormally safe. Add an IMI like imazethapyr (Pursuit) and the mixturebecomes-less safe (antagonized the crop safety). In the case of GAT Rd7,rimsulfuron causes crop phyto. Add an IMI like Pursuit and the mixturebecame more safe (antagonized the crop injury). The filed trialsdescribed below show there is an increased crop safety when normallyinjurious amounts of, for example, rimsulfuron were mixed with, forexample, imazethapyr, a normally “safe” imidazolinone herbicide. Example6A comprises a field trial that demonstrates this effect. Example 6Bprovides greenhouse data that confirms the field trial data.

Example 6A

The T6 generation of soybean of the lead GAT7 event (SEQ ID NO:68) alsohaving HRA (SEQ ID NO:65) [SCP:GAT7::SAMS:ALS] was compared to 92B25 todetermine levels of robustness when sprayed with different combinationsand rates of sulfonylurea, chlorpyrifos, and/or imazethapyr chemistries.Across all the treatment combinations except 16× Harmony® (nosignificance detected), GAT7 had significantly lower spray responsecompared to STS® at 7, 14, and 28 days after spraying (DAS). Applicationof 1× Resolve® with and without Lorsban® created a large response fromGAT7 and STS® at 7 DAS. The GAT7 was able to significantly recover fromthese treatments at 14 and 28 DAS, while the STS® line remained heavilydamaged. The GAT7 line had significantly less spray response to 4×Pursuit® compared to the STS® line at 7 DAS, while both lines were notstatistically different at 14 and 28 DAS. When 1× Resolve®+4× Pursuit®was applied, the GAT7 line had significantly higher tolerance comparedto the STS® line at 7, 14, and 28 DAS. At 7, 14, and 28 DAS 16×Harmony®, there was not a large response observed from either the GAT7or STS® line. Data from the treatments combining 16× Harmony® with 4×Pursuit® or 16× Harmony® with 4× Express® indicated GAT7 hadsignificantly lower spray response compared to the STS® line at 7, 14,and 28 DAS. In addition, the 7, 14, and 28 DAS data from 0.25×Resolve®+1.5× Express® treatment showed GAT7 had significantly highertolerance compared to STS®. In general, the data from this studyindicate GAT7 provides significantly higher tolerance compared to STS®across multiple herbicide and insecticide chemistries.

Materials and Methods

For each treatment, three replications of two 12 foot plots of the leadGAT7 event (EAFS 3560.4.3; GEID=JH12862528) and 92B25 (STS®) were grownin a RCB design (blocked by treatment) at two locations in Hawaii. Thenine different treatments applied at the V3 growth stage were; 1.1×Resolve® (2 oz) (35.0 g/ha a.i. rimsulfuron), 2.1× Resolve® (35.0 g/haa.i. rimsulfuron)+1× Lorsban® 4E (560.0 g/ha a.i. chlorpyrifos), 3. 4×Pursuit® (211.8 g/ha a.i. imazethapyr), 4.1× Resolve® (35.0 g/ha a.i.rimsulfuron)+4× Pursuit® (211.8 g/ha a.i. imazethapyr), 5.16× Harmony®GT (70.0 gms/ha a.i. thifensulfuron), 6.16× Harmony® (70.0 gms/ha a.i.thifensulfuron)+4× Pursuit® (211.8 g/ha a.i. imazethapyr), 7.16×Harmony® (70.0 gms/ha a.i. thifensulfuron)+4× Express® (35.0 g/ha a.i.tribenuron) 8.0.25× Resolve® (2,157 g/ha a.i. glyphosate)+1.5× Express®(13.1 g/ha a.i. tribenuron) 9. Unsprayed Control. All spray treatmentsalso contained a 1× non-ionic surfactant and ammonium sulfide. At 7, 14,and 28 days after spraying, plots were given a visual spray damagerating based upon observed chlorosis, necrosis, and/or plant stunting(0%=no observed effect to 100%=entire plot deceased). Visual rating datawere subject to ANOVA and mean separation using SAS.

Results and Discussion

Across all treatments at 7 and 14 DAS, the location, GEID, treatments,and GEID*treatment effects were significantly different (data notshown). The GAT7 line was scored significantly lower than the STS® lineacross all treatments at 7 and 14 DAS (data not shown). At 28 DAS, theGEID, loc*GEID, treatments, and GEID*treatment effects weresignificantly different (data not shown). Over all the treatments, theGAT7 line was scored with significantly less spray damage compared tothe STS® line at 28 DAS (data not shown).

The 1× Resolve® (rimsulfuron) treatment created a large response fromboth the GAT7 (70%); and STS® (83%) lines at 7 DAS (data not shown). At14 DAS, the GAT7 line (74%) had significantly less response compared tothe STS® line (93%) (data not shown). By 28 DAS, the GAT7 was able torecover and was scored with significantly less damage compared to theSTS® line (32% vs. 93%) (data not shown).

When date from the Lorsban® (chlorpyrifos)+1× Resolve® treatment areexamined, the GAT7 line did not have significantly less responsecompared to the response of the STS® line until 14 and 28 DAS (data notshown). At 28 DAS, the GAT7 line sprayed with Lorsban® and Resolve®(56%) did not recover as quickly compared to only the 1× Resolve® (32%)treatment on the GAT7 line (data not shown).

Application of 4× Pursuit® (imazethapyr) provided a large response fromthe STS® line (72%) at 7 DAS, while the GAT7 line (29%) hadsignificantly less response (data not shown). At 14 DAS and 28 DAS, thedifference between GAT7 and STS® was not significant (data not shown).

A tank mix of 1× Resolve®+4× Pursuit® resulted in significantly lessspray response for the GAT7 line compared to the STS® line at 7, 14, and28 DAS (data not shown). The 1× Resolve®+4× Pursuit® treatment on GAT7was scored with less damage overall at 7, 14, and 28 DAS compared to the1× Resolve® only and 1× Resolve®+Lorsban® treatment (data not shown).Using a pairwise comparison of these treatments on only the GAT7 line,the 1× Resolve®+4× Pursuit® treatment was scored significantly less thanthe 1× Resolve®+Lorsban® treatment at 14 and 28 DAS. In addition, apairwise comparison of the 1× Resolve®+4× Pursuit® treatment compared tothe 1× Resolve® treatment on GAT7 was only significantly less at 14 DAS.

Application of 16× Harmony® did not create a large response from theGAT7 or STS® lines at the three rating dates (data not shown). GAT7 wasnot significantly different from STS® for all the 16× Harmony® scores(data not shown). The treatment applying 4× Pursuit® mixed with 16×Harmony® did create significantly lower response scores for the GAT7line compared to the STS® line at 7, 14, and 28 DAS (data not shown).The mixture of 4× Express® with 16× Harmony® was scored significantlylower for the GAT7 line compared to the STS® line at 7, 14, and 28 DAS(data not shown). In general, the GAT7 line provided excellent overalltolerance to the 16× Harmony®, 16× Harmony®+4× Pursuit®, and 16×Harmony®+4× Express® treatments at all the scoring dates.

A mixture of 0.25× Resolve®+1.5× Express® created a crop response fromthe GAT7 line at 7 DAS (53%) and 14 DAS (47%) that was not as evident at28 DAS (12%) (data not shown). The STS® line had significantly higherresponse compared to GAT7 at 7 DAS (79%) and 14 DAS (90%) (data notshown). The STS® line did not recover at 28 DAS (87%) and hadsignificantly more response compared to the GAT7 line (data not shown).Examining only the GAT7 line, a pairwise comparison of the 0.25×Resolves+1.5× Express® treatment had significantly less responseobserved compared to the 1× Resolve® treatment at 7, 14, and 28 DAS.TABLE 19 Summary of Treatment Protocols TRT TREATMENT COMPONENTFORMULATION RATE UNIT TIMING 1 Resolve 2 oz A >DPX-E9636 (WG 25.00 PC)WG 25.00 PC 2.00 OMA 01 POSPOS B SURFACTANT - NON-IONIC (SL) SL 1.00 PR0.25 PMV 01 POSPOS C >AMSUL (GR 100 PC) GR 100.00 PC 2.00 LMA 01 POSPOS2 Resolve + Lorsban A >DPX-E9636 (WG 25.00 PC) WG 25.00 PC 2.00 OMA 01POSPOS B LORSBAN 4E (EC) EC 4.00 LG 1.00 PMA 01 POSPOS C SURFACTANT -NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01 POSPOS D >AMSUL (GR 100 PC) GR100.00 PC 2.00 LMA 01 POSPOS 3 Pursuit A PURSUIT DG (70WG) WG 70.00 PC4.32 OMA 01 POSPOS B SURFACTANT - NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01POSPOS C >AMSUL (GR 100 PC) GR 100.00 PC 2.00 LMA 01 POSPOS 4 Resolve +Pursuit A >DPX-E9636 (WG 25.00 PC) WG 25.00 PC 2.00 OMA 01 POSPOS BPURSUIT DG (70WG) WG 70.00 PC 4.32 OMA 01 POSPOS C SURFACTANT -NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01 POSPOS D >AMSUL (GR 100 PC) GR100.00 PC 2.00 LMA 01 POSPOS 5 Harmony GT 1.33 oz A >HARMONY GT PX (75%EXTRUDED WG 75.00 PC 1.33 OMA 01 POSPOS WG) B SURFACTANT - NON-IONIC(SL) SL 1.00 PR 0.25 PMV 01 POSPOS C >AMSUL (GR 100 PC) GR 100.00 PC2.00 LMA 01 POSPOS 6 GT + Pursuit A >HARMONY GT PX (75% EXTRUDED WG75.00 PC 1.33 OMA 01 POSPOS WG) B PURSUIT DG (70WG) WG 70.00 PC 2.16 OMA01 POSPOS C SURFACTANT - NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01 POSPOSD >AMSUL (GR 100 PC) GR 100.00 PC 2.00 LMA 01 POSPOS 7 Harmony GT 1.33 +EX 0.67 A >HARMONY GT PX (75% EXTRUDED WG 75.00 PC 1.33 OMA 01 POSPOSWG) B >EXPRESS PX (75% EXTRUDED WG) WG 75.00 PC 0.67 OMA 01 POSPOS CSURFACTANT - NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01 POSPOS D >AMSUL (GR100 PC) GR 100.00 PC 2.00 LMA 01 POSPOS 8 Resolve 0.5 + Express 0.25A >DPX-E9636 (WG 25.00 PC) WG 25.00 PC 0.50 OMA 01 POSPOS B >EXPRESS PX(75% EXTRUDED WG) WG 75.00 PC 0.25 OMA 01 POSPOS C SURFACTANT -NON-IONIC (SL) SL 1.00 PR 0.25 PMV 01 POSPOS D >AMSUL (GR 100 PC) GR100.00 PC 2.00 LMA 01 POSPOS 999 UNTREATED CHECK A UNTREATED CHECK NA0.00 NA 0.00 NA 00 UNTRCHK> = SUPPLEMENTAL CHEMICALRATE UNITS:LMA = POUNDS MATERIAL/ACRENA = NOT APPLICABLEOMA = OZ (DRY) MATERIAL/ACREPMA = PINTS MATERIAL/ACREPMV = % MATERIAL VOL TO VOLDESIGN: RANDOMIZED COMPLETE BLOCK DESIGNNO. REPS: 3PLOT SIZE: 5 × 10 FEETPLOT AREA: 50 SQUARE FEETOBSERVATIONS/RATING:Crop Response 7, 14 & 28 DATTIMINGS:00 = UNTRCHK, UNTREATED TIMING01 = POSPOS, POSTEMERGENCE V2-3

Example 6B

Example 6B provides greenhouse data that confirms the field trial dataprovided above in Example 6A. Soybeans comprising the lead GAT7 eventand also having HRA were used in the studies. TABLE 20 Summary oftreatment conditions Rimsulfuron Imazethapyr Rate Rate Replicates TestPlant (g ai/ha) (g ai/ha) Treatment # A B C D Mean GAT/HRA 0 0 1 53.9153.16 59.21 55.43 Soybeans 140 2 48.8 52.97 54.45 61.67 54.47 280 349.06 53.65 39.74 61.06 50.88 560 4 56.46 52.98 57.76 55.76 55.74 1024 551.34 53.89 45.64 51.47 50.59 35 0 6 21.09 20.45 27.11 20.83 22.37 140 718.18 18.79 26.03 22.15 21.29 280 8 26.45 15.59 14.53 26.5 20.77 560 922.2 22.97 26.8 20.65 23.16 1024 10 24.56 30.53 28.04 19.95 25.77 70 011 18.33 24.56 15.83 19.75 19.62 140 12 16.71 23.1 27.27 21.44 22.13 28013 27.43 34.8 32.26 21.65 29.04 560 14 28.84 26.17 28.12 31.95 28.771024 15 32.51 25.19 34.18 29.96 30.46 140 0 16 20.09 12.5 13.96 23.3217.47 140 17 21.69 24.38 16.15 17.27 19.87 280 18 23.77 21.72 27.1626.22 24.72 560 19 30.39 34.67 21.46 28.41 28.73 1024 20 35.19 30.68 2635.19 31.77 280 0 21 18.14 17.76 20.01 14.3 17.55 140 22 15.67 16.6218.29 15.91 16.62 280 23 15.83 21.36 20.3 24.67 20.52 560 24 23.9 19.2228.67 24.7 24.12 1024 25 30.93 28.76 24.69 36.65 30.26 Jack 0 0 1 50.9360.34 53.87 46.89 53.01 Soybeans 70 2 45.88 42.49 41.6 37.47 41.86 140 338.09 38.57 28.06 42.33 36.76 280 4 35.25 33.01 42.97 41.4 38.16 560 539.2 37.15 38.6 29.67 36.16 0.5 0 6 26.27 18.24 21.48 16.09 20.52 70 715.12 13.93 11.01 7.12 11.80 140 8 12.68 6.92 10.82 12.51 10.73 280 912.86 7.14 12.16 9.61 10.44 560 10 12.74 10.68 12.71 11.06 11.80 1 0 115.8 7.64 7.13 9.21 7.45 70 12 8.05 6.76 8.42 7.48 7.68 140 13 7.57 6.547.49 10 7.90 280 14 6.84 7.92 7.25 8.37 7.60 560 15 12.51 10.59 9.577.16 9.96 2 0 16 4.09 5.37 4.3 11.58 6.34 70 17 3.36 6.57 4.83 7.67 5.61140 18 6.12 6.55 5.59 6.09 6.09 280 19 5.87 4.89 5.48 6.24 5.62 560 205.35 7.73 8.9 5.48 6.87 4 0 21 4.12 4.48 2.93 3.46 3.75 70 22 2.75 2.9411.64 4.21 5.39 140 23 3.29 3.23 4.24 3.59 280 24 4.46 5.9 3.92 3.654.48 560 25 4.67 5.01 2.77 4.1 4.14

TABLE 21 Summary of greenhouse results. Mean- RimsulfuronRateImazethapyr Replicates Standard Initial % Growth Test Plant (g ai/ha)Rate (g ai/ha) Treatment # A B C D Mean Deviation Weight ReductionGAT/HRA 0 0 1 53.91 53.16 59.21 55.43 3.30 49.28 0 Soybeans 140 2 48.852.97 54.45 61.67 54.47 5.36 48.33 2 280 3 49.06 53.65 39.74 61.06 50.888.92 44.73 9 560 4 56.46 52.98 57.76 55.76 55.74 2.02 49.60 −1 1024 551.34 53.89 45.64 51.47 50.59 3.50 44.44 10 35 0 6 21.09 20.45 27.1120.83 22.37 3.17 16.23 67 140 7 18.18 18.79 26.03 22.15 21.29 3.61 15.1469 280 8 26.45 15.59 14.53 26.5 20.77 6.60 14.62 70 560 9 22.2 22.9726.8 20.65 23.16 2.61 17.01 65 1024 10 24.56 30.53 28.04 19.95 25.774.59 19.63 60 70 0 11 18.33 24.56 15.83 19.75 19.62 3.67 13.47 73 140 1216.71 23.1 27.27 21.44 22.13 4.37 15.99 68 280 13 27.43 34.8 32.26 21.6529.04 5.80 22.89 54 560 14 28.84 26.17 28.12 31.95 28.77 2.40 22.63 541024 15 32.51 25.19 34.18 29.96 30.46 3.92 24.32 51 140 0 16 20.09 12.513.96 23.32 17.47 5.10 11.32 77 140 17 21.69 24.38 16.15 17.27 19.873.84 13.73 72 280 18 23.77 21.72 27.16 26.22 24.72 2.46 18.57 62 560 1930.39 34.67 21.46 28.41 28.73 5.51 22.59 54 1024 20 35.19 30.68 26 35.1931.77 4.39 25.62 48 280 0 21 18.14 17.76 20.01 14.3 17.55 2.38 11.41 77140 22 15.67 16.62 18.29 15.91 16.62 1.18 10.48 79 280 23 15.83 21.3620.3 24.57 20.52 3.61 14.37 71 560 24 23.9 19.22 28.67 24.7 24.12 3.8817.98 64 1024 25 30.93 28.75 24.69 36.65 30.26 4.99 24.11 51

Example 7 Methods of Transformation Employing a GAT Sequence in Maize

I. Preparation of Agrobacterium Master Plate

1. Obtain engineered Agrobacterium tumefaciens strain with GATcomponents (SEQ ID NO: 70 or SEQ ID NO:55) and stored in −80° C. degreefreezer as a 50% glycerol stock. The transcriptional control region usedwas the 3×35S ENH (−) operably linked to the ZmUbi PRO-5UTR-ZmUbi intron1 promoter (SEQ ID NO:78). This transcriptional control region (SEQ IDNO:78) is set forth below denoting the location of the various regionsof the regulatory region: a) the 35S enhancer (3×) in the reversedirection has a single underline; b) the UBI promoter has a doubleunderline, and c) the UBI intron is in italics. (SEQ ID NO:78)atcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatggacggtatcgataagctagcttgatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatgatcgaattatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatgggaattcctgcagcccagcttgcatgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactcctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctcccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca

-   2. Prepare master plate from a glycerol stock by streaking the    bacteria to produce single colonies on #800 medium and incubate the    bacteria at 28° C. in the dark for 3-4 days.-   3. Prepare a working plate by streaking 1 colony from the master    plate across #810 media. Incubate bacteria at 28° C. in the dark for    1-2 days.    II. Preparation of Bacteria for Embryo Infection    -   1. Prepare liquid culture of Agrobacterium 1 day prior to embryo        isolation. Set up a flask with 30 mls of 557A medium, 30 μl of        2% acetosyringone and 30 μl of 5% spectinomycin.    -   2. Inoculate with 1 loopful of Agrobacterium from 810 medium and        place on shaker (200 rpm) in dark room at 28° C. overnight.    -   3. On morning of infection, take samples of the liquid culture        of Agrobacterium and make a ¼ dilution with 557A. Use the        diluted liquid culture to take OD reading using visible light at        550 nm.    -   4. Make dilutions to Agrobacterium culture as appropriate        according the OD reading to 30 maintain OD reading between        0.2-0.8 during embryo isolation.    -   5. When preparing Agrobacterium for infection, repeat OD reading        of liquid culture. Using the OD reading calculate the number of        mls required to obtain 5 E10 cfu/ml (cfu=colony forming unit) by        using the formula EXPONENT (1.755*(InOD)+21.77) as derived from        a standard curve. Pipet the calculated amount of Agrobacterium        liquid culture into 14 ml tube and centrifuge at 4500 rpm at        4-20° C. for ten minutes. Remove the supernatant and resuspend        Agrobacterium in appropriate amount of 100 uM acetosyringone        solution in 561Q.        III. Immature Embryo Isolation    -   1. Harvest GS3 ears at 9-11 days after pollination with embryo        size of 1-2 mm in length.    -   2. Sterilize ear in 50% bleach and 1 drop Tween for 20-30        minutes. Rinse 3-5 times in sterile water    -   3. Isolate embryos from kernels and place in microtube        containing 2 mls 561Q.        VI. Agrobacterium Infection of Embryos    -   1. Remove 561Q with pipette from the microtube with isolated        embryos and add 1 ml of Agrobacterium suspension at OD described        above.    -   2. Mix by vortexing for about 30 seconds.    -   3. Allow 5 minutes for infection at room temperature.        V. Co-Cultivation    -   1. After removing liquid medium, transfer embryos and orient the        embryos with embryonic axis down on the surface of 562P        co-cultivation medium.    -   2. Place embryos in 20° C. incubator for 3 days. Transfer to        28° C. for 3 additional days.        VI. Selection of Transgenic Putative Callus Events    -   1. After co-cultivation, transfer embryos to 5631 selection        medium containing 1 mM glyphosate. Culture the embryos at 28° C.        in dark.    -   2. Every 14-21 days transfer embryos to fresh 563I medium. The        selection process may last about 2 months until actively growing        putative callus events can be identified. Maintain putative        callus events on 563I medium and sample callus for PCR.        VII. Regeneration of T0 Plants    -   1. Transfer callus events to 287I medium containing 0.1 mM        Glyphosate until somatic embryos mature. Culture the callus at        28° C. in dark.    -   2. Transfer mature embryos to 273I embryo germination medium        containing 0.1 mM glyphosate in plates. Culture the plates at        28° C. in light.    -   3. When shoots and roots emerge, transfer individual plants to        273I containing 0.1 mM Glyphosate in tubes. Culture the tubes at        28° C. in light.    -   4. Plantlets with established shoots and roots shall be        transferred to greenhouse for further growth and production of        T1 seed.

Example 8 Effect of 35S Enhancer on Transformation Efficiency andEfficacy of GAT and ALS in Maize

Materials and Methods

Four 35S enhancer constructs (PHP20118, PHP20120, PHP20122, PHP20124)and one non-35S construct (PHP19288) were used to produce events toevaluate the effect of 35S enhancer on transformation efficiency andefficacy of GAT (SEQ ID NO:70) (FIG. 1). The differences between thefour 35S enhancer constructs are the copy numbers of the 35S enhancerand the orientations of the 35S enhancer in the constructs. A summary ofeach 35S enhancer construct is provided below.

PHP20118 comprises 35S ENH(+):ZmUBI PRO-5UTR-UBI INTRON1 (+denotesforward direction of 35S enhancer). This transcriptional control region(SEQ ID NO:80) is set forth below denoting the location of the variousregions of the regulatory region: a) the 35S enhancer in the forwarddirection has a single underline; b) the UBI promoter has a doubleunderline, and c) the UBI intron is in italics. (SEQ ID NO:80)cccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatcaagcttatcgataccgtcgacctcgagggggggcccagcttgcatgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgtttctcctttttttttgcaaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctcccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca

PHP20122 comprises 3×35S ENH (+):ZmUBI PRO-5UTR-UBI INTRON1 (+denotesforward direction of 35S enhancer). This transcriptional control region(SEQ ID NO:81) is set forth below denoting the location of the variousregions of the regulatory region: a) the 35S enhancer in the forwarddirection has a single underline; b) the UBI promoter has a doubleunderline, and c) the UBI intron is in italics. (SEQ ID NO: 81)cccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgataattcgatcatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatcaagcttatcgataccgccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatgtctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctcccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca

PHP20120 comprises 35S ENH (−):ZmUBI PRO-5UTR-UBI INTRON1 (−denotesreverse direction of 35S enhancer). This transcriptional control region(SEQ ID NO:82) is set forth below denoting the location of the variousregions of the regulatory region: a) the 35S enhancer in the reversedirection has a single underline; b) the UBI promoter has a doubleunderline, and c) the UBI intron is in italics. (SEQ ID NO:82)atcacatcaatccacttgctttgaagacgtggttggaacgtcttcttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatgggaattcctgcagcccagcttgcatgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttcgcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctcccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcacttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca

PHP20124 comprises 3×35S ENH (−): ZmUBI PRO-5UTR-UBI INTRON1 (−denotesreverse direction of 35S enhancer). This transcriptional control region(SEQ ID NO:83) is set forth below denoting the location of the variousregions of the regulatory region: a) the 35S enhancer in the reversedirection has a single underline; b) the UBI promoter has a doubleunderline, and c) the UBI intron is in italics. (SEQ ID NO:83)atcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctgtatttgaatctttgactccatggacggtatcgataagctagcttgatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatgatcgaattatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaatgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagcgtgtcgtgctccaccatgttgacgaagattttcttcttgtcattgagtcgtaagagactctgtatgaactgttcgccagtctttacggcgagttctgttaggtcctctatttgaatctttgactccatgggaattcctgcagcccagcttgcatgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccgcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattccttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctcccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgCatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtcgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca

The transformation experiments were conducted side-by-side using thesame embryos from the same ears. Immature embryos of GS3 line wereaseptically removed from each ear and divided into five portions. Eachportion of the embryos was then infected with A. tumefaciens strainLBA4404 containing the expression cassettes from each of the fiveconstructs, respectively. After 6 days co-cultivation, the embryos weretransferred to fresh selection medium containing glyphosate. Thetransformed cells, which survived the glyphosate selection, proliferatedand produced somatic embryogenic calli. After about two monthssubculture, the calli were then manipulated to regenerate wholetransgenic plants with glyphosate presence and were transferred to thegreenhouse. T0 plants were then subjected to glyphosate spray at 6× (156oz/ac) Roundup Ready UltraMax™at V3 or V4 stage in the greenhouse.Positive plants were sampled for quantitative PCR for copy number andwestern for expression. T0 plants were then crossed with inbred lines toobtain seeds for further evaluation. Results

Transformation efficiency was measured as the percentage of the infectedembryos that produced resistant calli after selection. The averagetransformation efficiencies for PHP19288, PHP20118, PHP20120, PHP20122,and PHP20124 were 58%, 63%, 59%, 57%, and 51%, respectively. The dataindicated that all constructs had quite high and similar transformationefficiencies, although PHP20118 showed a slight increase (FIG. 3).

T0 plant efficacy was defined as the percentage of the T0 events thatwere completely resistant to the 6× glyphosate spray. The efficacy ofthe non-35S construct (PHP19288) was 48.1%. In contrast, the efficaciesof the 35S enhancer constructs (PHP20118, PHP20120, PHP20122, andPHP20124) were 96.6%, 93.5%, 89.1%, and 91.1%, respectively (FIG. 4).The data showed that all 35S enhancer constructs significantly increasedthe plant efficacy against glyphosate.

Another significant improvement of using 35S enhancer was in integrationpattern of the transgene. The percentage of the tested events that weresingle copy for the non-35S enhancer construct was only 38%, but for thefour 35S enhancer constructs (PHP20118, PHP20120, PHP20122, andPHP20124) single copy events represented 65%, 63%, 71%, and 88% of theevents, respectively (FIG. 4).

A subset of events from all five constructs were sampled by Westernanalysis to look any comparative differences in GAT expression betweennon 35S and 35S events. This analysis showed that events from thenon-35S enhancer construct had very low levels of GAT expression whereasthe majority of the events from the 35S enhancer constructs showed veryhigh levels of GAT expression (FIG. 5).

Example 9 Using 35S Enhancer GAT in Developing a Novel Callus-BasedGene/Construct Evaluation System

Materials and Methods

This assay is being developed to improve the evaluation of expression ofan insecticidal gene at a very early stage in the transformation processin order to identify potential problems with expression. The basis ofthis assay is the use of the glyphosate acetyl transferase (GAT) gene(SEQ ID NO:55) as a selectable marker. Both GAT and the insecticidaltest gene will be driven by a strong constitutive promoter and linked inthe same construct. The promoter employed comprised the ZmUBIPRO-5UTR-UBI INTRON1 with the 3×35S enhancer as described above inExample 7. As a result it is expected that selection on high levels ofglyphosate will identify high insecticidal test gene expressors. Thecallus tissue from these putative high expressors will then be used ininsect bioassays to determine whether the gene product is functional.Those constructs showing efficacy can be advanced into transformation.If the construct does not show efficacy then follow up biochemical andmolecular analyses can be conducted to identify the problem and the genewill be redesigned and retested in the system (FIG. 6).

Results

The assay is currently under development. Preliminary data has shownthat the activity of an efficacious insect control gene can be detectedat the callus stage. The correlation between the callus activity and theplant efficacy is currently being evaluated.

Example 10 GAT as a Selectable Marker

Materials and Method

Agrobacterium mediated transformation was used to introduce the GA T(SEQ ID NO:55) expression cassette into the corn genome. The GATexpression cassette comprises, promoter comprising ZmUBI PRO-5UTR-UBIINTRON1 with the 3×35S enhancer (as described above in Example 1)operably linked to the gat gene, and pinII terminator. Agrobacteriumtumefaciens, strain LBA4404, was pathogenically disarmed by removing itsnative T-DNA. Instead, the T-DNA site on the Ti plasmid contained theGAT expression cassette.

Immature embryos of maize were aseptically removed from the developingcaryopsis and treated with A. tumefaciens strain LBA4404 containing GATexpression cassettes. After a period of embryo and Agrobacteriumco-cultivation on solid culture medium without glyphosate presence, theembryos were transferred to fresh selection medium that containedantibiotics and glyphosate. The antibiotics kill any remainingAgrobacterium. The selection medium is stimulatory to maize somaticembryogenesis and selective for those cells that contain an integratedgat gene. Therefore, callus that survives glyphosate to proliferate andproduce embryogenic tissue is presumably genetically transformed. Callussamples were taken for molecular analysis to verify the presence of thetransgene by PCR. The embryonic tissue is then manipulated to regeneratetransgenic plants in the presence of glyphosate that are thentransferred to the greenhouse. T0 plants are sprayed with glyphosate atdifferent concentrations. Positive plants are sampled for molecularanalysis for transgene copy number and crossed with inbred lines toobtain seeds from the initially transformed plants.

A glyphosate kill curve was established by testing non-transformedembryos response on media with different levels of glyphosate. GS3embryos were isolated from an immature ear and placed onto mediacontaining glyphosate at 0.0, 0.5, 1.0, and 2.0 mM. After about 40 daysculture, the response of the embryos were observed and recorded.Similarly, infected GS3 embryos with the GAT construct were placed ontomedia containing glyphosate at 0.0, 0.5, 1.0, and 2.0 mM. After about 40days culture, the response of the infected embryos were observed andrecorded (FIG. 7).

A side-by-side experiment was conducted to compare the transformationefficiencies of GAT, bar and mopat. Immature embryos of GS3 line wereaseptically removed from each ear and divided into three portions. Eachportion of the embryos was then infected with A. tumefaciens strainLBA4404 containing the expression cassettes of GAT, bar, or mopatrespectively. After co-cultivation, the embryos infected with GATconstruct were selected on routine glyphosate medium and the embryosinfected with bar or mopat constructs were selected on routineglufosinate medium. The subcultures were done every 2 weeks. At about 50days selection the responses of the embryos were observed and recorded.

Results

From the glyphosate kill curve experiment, all embryos on medium with0.0 mM glyphosate initiated healthy callus, while about half of theembryos on medium with 0.5 mM glyphosate showed callus initiation. Therewas very little callus growth with embryos on media containing 1.0 and2.0 mm glyphosate. This indicated that 0.5 mM is not enough to inhibitall embryos growth, but 1 mM or 2 mM is strong enough to kill thenon-transformed embryos. In the infected embryo experiment, more calluswas grown on media with 0.0 and 0.5 mM glyphosate, but some embryosinitiated resistant callus on media with 1.0 mM or 2.0 mM glyphosate.Western or PCR analysis has confirmed that these resistant calli weretransformed. Currently, GAT has performed consistently as an effectiveselectable marker with excellent transformation efficiency in both GS3and introEF09B genotypes (FIG. 10 and Table 45). TABLE 22 GATtransformation efficiency in introEF09B txn % based on # selectable #infected # events events to genotype construct marker embryos to GH GHEFWWBTX GATHRA GAT 1332 354 27% EFWWCTX GATHRA GAT 136 47 35% EFWWETXGATHRA GAT 1109 158 14% EFWWZTX GATHRA GAT 1790 502 28% 4367 1061 24%In the side-by-side experiment to compare GAT, bar and mopat, GAT gavethe best transformation efficiency at about 64%, bar at 34%, and mopatat 30%. Calli with GAT selection seem to grow faster that those selectedon glufosinate (FIG. 8).

Example 11 Interaction Between Glyphosate, Metsulfuron-Methyl and TwoAdditives on the Control of Ryegrass

The example was conducted on non-glyphosate resistant ryegrass. Asprovided in Table 23, all treatments that included glyphosate [180 ga.i. L ha⁻¹ (0.5 L glyphosate (SCAT®))] plus metsulfuron-methyl(BRUSH-OFF®) (5 and 10 g ha⁻¹) gave 100% control of the ryegrass.Metsulfuron-methyl (BRUSH-OFF®) on its own did nothing accept stunt theryegrass plants slightly. Although 15 of the 16 plants (12%) in the 0.5L ha⁻¹ glyphosate (SCAT®) treatment eventually died, they took muchlonger to die. These results indicate that there was possible synergismbetween glyphosate (SCAT®) and metsulfuron-methyl (BRUSH-OFF®). The twoadditives, e.g., ADD-UP and VELOCITY, made no difference in the degreeof control with any of the treatments. TABLE 23 Interaction BetweenGlyphosate, metsulfuron-methyl and Two Additives on the Control ofRyegrass Control Trial Treatment/ha⁻¹ (%) 1 glyphosate (SCAT ®) 0.5 L 942 Metsulfuron-methyl (BRUSH-OFF ®) 5 g 0 3 Metsulfuron-methyl(BRUSH-OFF ®) 10 g 0 4 glyphosate (SCAT ®) 0.5 L + 100metsulfuron-methyl (BRUSH-OFF ®) 5 g 5 glyphosate (SCAT ®) 0.5 L + 100metsulfuron-methyl (BRUSH-OFF ®) 10 g 6 glyphosate (SCAT ®) 0.5 L + 100metsulfuron-methyl (BRUSH-OFF ®) 5 g + ammonium sulfate-based adjuvant(ADD-UP ®) 1% 7 glyphosate (SCAT ®) 0.5 L + 100 metsulfuron-methyl(BRUSH-OFF ®) 10 g + ammonium sulfate-based adjuvant (ADD-UP ®) 1% 8glyphosate (SCAT ®) 0.5 L + 100 metsulfuron-methyl (BRUSH-OFF ®) 5 g +bispyribac-sodium (VELOCITY ®) 1% 9 glyphosate (SCAT ®) 0.5 L + 100metsulfuron-methyl (BRUSH-OFF ®) 10 g + bispyribac- sodium (VELOCITY ®)1% 10  Control 0 Biotype: non-glyphosate resistant ryegrass (70-04)Population profile: Rup 0.25 control (%) 6.25 Rup 0.5 control (%) 81.25Rup 1.0 control (%) 93.75 Growth Stage: 5-6 leaf stage Conditions: hotand sunny Volume rate: 200 L ha⁻¹

Example 12 Interaction Between Glyphosate, metsulfuron-methyl and TwoAdditives on the Control of Hairy Fleabane (Conyza bonariensis)

This example included the same treatments as used in Example 11, exceptthe treatments were carried out on a glyphosate sensitive biotype ofKleinskraalhans (Conyza bonariensis). Glyphosate on its own gave poorcontrol, killing only 1 out of 16 plants (12%). In this example,however, ammonium sulfate-based adjuvant (ADD-UP®) performed better thanbispyribac-sodium (VELOCITY®) with the mixture. TABLE 24 InteractionBetween Glyphosate, metsulfuron-methyl and Two Additives on the Controlof Hairy Fleabane (Conyza bonariensis) Trial Treatment/ha⁻¹ Control (%)1 glyphosate (ROUNDUP ®) 0.5 L 12 2 Metsulfuron-methyl (BRUSH-OFF ®) 5 g100 3 Metsulfuron-methyl (BRUSH-OFF ®) 10 g 100 4 glyphosate (ROUNDUP ®)0.5 L + metsulfuron- 100 methyl (BRUSH-OFF ®) 5 g 5 glyphosate(ROUNDUP ®) 0.5 L + metsulfuron- 100 methyl (BRUSH-OFF ®) 10 g 6glyphosate (ROUNDUP ®) 0.5 L + metsulfuron-methyl (BRUSH- 100 OFF ®) 5g + ammonium sulfate-based adjuvant (ADD-UP ®) 1% 7 glyphosate(ROUNDUP ®) 0.5 L + metsulfuron-methyl (BRUSH- 100 OFF ®) 10 g +ammonium sulfate-based adjuvant (ADD-UP ®) 1% 8 glyphosate (ROUNDUP ®)0.5 L + metsulfuron-methyl (BRUSH- 75 OFF ®) 5 g + bispyribac-sodium(VELOCITY ®) 1% 9 glyphosate (ROUNDUP ®) 0.5 L + metsulfuron-methyl(BRUSH- 100 OFF ®) 10 g + bispyribac-sodium (VELOCITY ®) 1% 10 Control 0Biotype: Non-glyphosate resistant Conyza (Welgevallen-paraquatresistant)Growth Stage: 10-15 leaf stageConditions: Cool and sunnyVolume rate: 200 L ha⁻¹glyphosate (ROUNDUP): 360 g a.i. L⁻¹

Example 13 Interaction Between Glyphosate, Metsulfuron-Methyl and TwoAdditives on the Control of Glyphosate, Paraquat, and ACCase-InhibitorResistant Ryegrass

This example was conducted on one of the most resistant ryegrass typesin the world, ryegrass resistant to non-selective herbicides, e.g.,Fairview (Tulbagh), which is resistant to glyphosate, paraquat, andACCase-inhibitors. In this example, the addition of metsulfuron-methyl(BRUSH-OFF®) at 5 and 10 g ha⁻¹, with 1% ammonium sulfate-based adjuvant(ADD-UP®) to 0.5 L Roundup improved control by 44% (e.g., 50% to 94%).Further, ammonium sulfate-based adjuvant (ADD-UP®) was superior tobispyribac-sodium (VELOCITY®) as an additive. TABLE 25 InteractionBetween Glyphosate, metsulfuron-methyl and Two Additives on the Controlof Glyphosate, Paraquat, and ACCase-Inhibitor Resistant Ryegrass TrialTreatment/ha⁻¹ Control (%) 1 glyphosate (ROUNDUP ®) 0.5 L 50 2glyphosate (ROUNDUP ®) 0.5 L + metsulfuron- 69 methyl (BRUSH-OFF ®) 5 g3 glyphosate (ROUNDUP ®) 0.5 L + metsulfuron- 88 methyl (BRUSH-OFF ®) 10g 4 glyphosate (ROUNDUP ®) 0.5 L + metsulfuron-methyl 94 (BRUSH-OFF ®) 5g + ammonium sulfate-based adjuvant (ADD- UP ®) 1% 5 glyphosate(ROUNDUP ®) 0.5 L + metsulfuron-methyl 94 (BRUSH-OFF ®) 10 g + ammoniumsulfate-based adjuvant (ADD-UP ®) 1% 6 glyphosate (ROUNDUP ®) 0.5 L +metsulfuron-methyl 69 (BRUSH-OFF ®) 5 g + bispyribac-sodium (VELOCITY ®)1% 7 glyphosate (ROUNDUP ®) 0.5 L + metsulfuron-methyl 75 (BRUSH-OFF ®)10 g + bispyribac-sodium (VELOCITY ®) 1%Biotype: Fairview (Tulbagh) resistant to glyphosate, paraquat, andACCase-inhibitors.Growth Stage: 10-15 leaf stageConditions: Cool and sunnyVolume rate: 200 L ha⁻¹glyphosate (ROUNDUP): 360 g ai L⁻¹

Example 14 Interaction Between Glyphosate and Representative SU's on theControl of Glyphosate Paraquat and ACCase-Inhibitor Resistant Ryegrass

In this example, four different SU's were applied together withglyphosate (SCAT®) on the resistant ryegrass. The results as to the bestSU partner for glyphosate were inconclusive, but the average benefit ofapplying an SU with glyphosate on herbicide resistant ryegrass was 39%(e.g., 34% control with glyphosate (SCAT®) only and 57-83% control withglyphosate (SCAT®) plus metsulfuron-methyl (BRUSH-OFF®), chlorsulfuron(GLEAN®), or triasulfuron (LOGRAN(®). TABLE 26 Interaction BetweenGlyphosate and Representative SU's on the Control of Glyphosate,Paraquat, and ACCase-Inhibitor Resistant Ryegrass Control TrialTreatment/ha⁻¹ (%) 1 Control 0 2 glyphosate (SCAT ®) 6 L 34 3 glyphosate(SCAT ®) 6 L + metsulfuron-methyl 67 (BRUSH-OFF ®) 10 g 4 glyphosate(SCAT ®) 6 L + chlorsulfuron 75 (GLEAN ®) 15 g 5 glyphosate (SCAT ®) 6L + triasulfuron 83 (LOGRAN ®) 7½ g 6 glyphosate (SCAT ®) 6 L +triasulfuron 67 (LOGRAN ®) 15 gBiotype: Fairview (Tulbagh) resistant to glyphosate, paraquat, andACCase-inhibitors.Growth Stage: 4 leaf stageConditions: Cool and sunnyVolume rate: 200 L ha⁻¹All treatments sprayed with 1% ADD-UP

Example 15 GAT Has No Yield Impact on Soybean Isolines

Abstract

Isolines from twelve selected SCP:GAT7::SAMS:ALS events were yieldtested in 3 Iowa environments in 2004 and 6 midwest environments in2005. When yield data for these environments is combined together, thereis no significant yield difference between GAT7 positive lines and GAT7negative lines across the construct, and within a specific event. Forthe three lead events (EAFS 3559.2.1, EAFS 3560.4.3, EAFS 3561.1.1),there were no statistical yield differences detected when GAT7 positivelines were compared to GAT7 negative sister lines. Overall, the data forthe lines tested indicate that presence of the GAT7 transgene does notappear to impact final yield.

Materials and Methods

2004 D Test Materials and Methods:

The variety Jack was transformed with the constituative promoter (SCP1)driving expression of glyphosate acetyl transferase round 7 (GAT7),linked to the selectable marker insert SAMS:ALS. Forty initial events ofSCP:GAT7::SAMS:ALS were advanced to the T2 generation. Zygosity of theadvanced T2 lines was initially determined by screening 12 random plantsper line for PCR amplification of the GAT insert. 454 lines weretentatively selected to be either homozygous positive or homozygousnegative for GAT. Selected lines were blocked by event and grown in Dlevel yield tests (1 replication of two 10 foot rows) at Cedar Falls(planted Jun. 5, 2004), Dallas Center (planted Jun. 4, 2004), andJohnston, Iowa (planted Jun. 9, 2004) during 2004. Twelve remnant T3seed of each line was screened using either glyphosate spray at the V3growth stage, or soaking remnant seed in sulfonylurea solution. Basedupon the glyphosate treatment or SU seed soak, 342 SCP:GAT7::ALS linesfrom 30 events were confirmed to be homozygous positive or homozygousnegative. Maturity scores were collected for all entries at the DallasCenter and Johnston locations. Yield data was collected and subject tomultiple regression, ANOVA, and mean separation using SAS.

2005 C Test Materials and Methods

Based upon yield performance and herbicide efficacy scores in 2004, 12GAT7 events were advanced for C level yield testing in 2005. From theseselected twelve events, 28 positive and 23 negative isolines wereselected. 2005 C tests were designed as a randomized complete block (byevent) and grown at Cedar Falls, Iowa; Johnston, Iowa; Stuart, Iowa;Monmouth, Ill.; Princeton, Ill.; and Napoleon, Ohio. Maturity scores andyield data were collected and subject to multiple regression, ANOVA andmean separation using PRISM and/or SAS.

Results and Discussion

When 2004 yield data for the selected lines tested was subject to ANOVA,the location, events, location*event, and GAT (positive or negative)variables were significantly different (data not shown). A mean of allpositive lines was not significantly lower for yield compared to a meanof all negative lines tested in 2004. In 2005, the location, event,location*event, GAT, and GAT*location variables were significantlydifferent (data not shown). A mean of all positive lines tested was notsignificantly different from a mean of all negative lines tested in 2005(data not shown). When the 2004 and 2005 data are combined, the year,location, event, year*event, location*event, year*GAT, and location*GATvariables are significantly different (data not shown). A mean of allGAT positive lines was not significantly different from a mean of allGAT negative lines tested across the 9 midwest environments (data notshown).

Based upon 2004 yield, herbicide efficacy, and molecular analyses, 3lead GAT7 events were selected for potential regulatory and productdevelopment experiments. When the 2005 isoline yield data is combinedwith 2004 data, the locations were significantly different within EAFS3559.2.1 (data not shown). Positive GAT lines within EAFS 3559.2.1 werenot significantly different for yield compared to negative sisterisolines (data not shown). Within EAFS 3560.4.3, the 9 locations andlocation*GAT interaction were significantly different. However, GATpositive isolines were not significantly different for yield whencompared to GAT negative isolines within the same event (data notshown). Within event EAFS 3561.1.1, the locations were significantlydifferent, while the GAT score and location*GAT interaction were notsignificantly different (data not shown). Within EAFS 3561.1.1, GATpositive lines were not significantly different for yield compared toGAT negative sister lines (data not shown).

Multiple regression of yield×maturity for the 6 environments tested in2005 was performed on the 3 lead events to determine overall yieldpotential. In general, GAT positive and GAT negative lines within eachof the 3 lead events appear to be random (data not shown). This suggeststhat yield does not appear to be significantly altered when the GAT7transgene is present.

A modified t-test was completed to compare the GAT7 positive lines to amean of the GAT7 negative lines within each specific event at eachlocation tested (data not shown). Across the 9 locations tested, thereis no apparent yield disadvantage for GAT7 positive lines compared toGAT7 negative sister lines within the same event. For the 3 lead events,GAT positive lines are within 2.6% of the negative mean, indicatingoverall yield parity exists in all environments tested (data not shown).ANOVA and LSD analyses performed on the individual lines indicate nodistinct differences among lines tested in each event, except for EAFS3560.3.2 (data not shown). Overall, there does not appear to be a yielddifference between GAT7 positive lines and GAT7 negative lines.

Example 16 GAT Soybeans Have Tolerance to Glyphosate and Glyphosate+SUTreatments

The objectives of this experiment was to evaluate the sulfonylurea (SU)herbicide tolerance of the lead GAT7 events in direct comparison to thetolerance of STS, and to determine if differences in tolerance could bedetected among the lead GAT7 events across different glyphosate (Gly),Gly+SU and SU treatments. Across all the treatments, the four lead GAT7events were rated with significantly less crop damage response comparedto untransformed Jack and STS at 7 days after spraying, and again at 14days after spraying. Among the 4 lead GAT7 events, there were someresponse differences detected, with EAFS 3560.4.3 performing the bestover all the treatments, and EAFS 3560.3.2 showing the most herbicideresponse. In general, it appears there is a significantly bettertolerance to several SU chemistries for the SAMS:ALS construct comparedto STS. In addition, the GAT7 events tested had good tolerance tovariable rates of glyphosate, sulfonylurea, and glyphosate plussulfonylurea chemistry treatments.

Materials and Methods

GAT round 7 (GAT7) transgenic events from construct PHP20163A wereevaluated in 2004 for herbicide efficacy (JHM464TGATEFF) and yieldpotential (JHD4_GAT7 tests). Based upon these preliminary results andcommercialization potential, four lead events were selected foradditional efficacy testing in 2005 experiment JHM5G030.92M90 wasselected as a STS variety to compare directly with the SAMS:ALSconstruct in the GAT7 events. Variety Jack was utilized as anuntransformed negative control.

Selected lines were grown in experiment JHM5G030 in two replications ofpaired twelve foot rows, with eleven different treatments applied. Lineswere blocked by event and treatment to provide side-by-side comparisonof the eleven different spray treatments, with a 2 row border betweeneach treatment to catch any spray drift. Spray treatments (at V3 unlessspecified) were OX (control), 35.03 g/ha ai Synchrony, 140.11 g/ha aiSynchrony, 8.75 g/ha ai tribenuron, 35.0 g/ha ai tribenuron, 8.75 g/haai rimsulfuron, 35.0 g/ha ai rimsulfuron, 3360.0 g/ha ai glyphosate,3360.0 g/ha ai glyphosate at V3 followed by 3360.0 g/ha ai glyphosate atR1, 3360.0 g/ha ai glyphosate plus 35.0 g/ha ai tribenuron (tank mix)and 3360.0 g/ha ai glyphosate plus 70.0 g/ha ai rimsulfuron (tank mix).Lines were given a 100 (complete susceptibility, 100% damage) to 0(complete tolerance, 0% damage) visual rating at 7 days after treatmentand again at 14 days after treatment. Visual ratings were based uponoverall degree of chlorosis, necrosis, and plant stunting (if evident)in the treated rows compared to the respective unsprayed control rows.Rating data at 7 and 14 days after spraying were subject to ANOVA andmean separation using SAS.

Results and Discussion

When all spray rating data for the different treatments at 7 and 14 daysafter application were subject to ANOVA, the events and treatments weresignificantly different, while the replication was not significantlydifferent (data not shown). Jack had significantly higher spray response(damage scores) compared to STS and the GAT events at 7 and 14 daysafter spraying (DAS) (data not shown). The 4 GAT lines were scored withsignificantly less damage than Jack and STS for all the treatments atboth 7 and 14 days after spray application (data not shown).

In examining individual treatments, the control plot appeared to havesome spray drift evident, as both the Jack plot and STS plot were ratedwith significantly higher spray damage scores compared to the 4 GATevents at 7 DAS (data not shown) and 14 DAS (data not shown). Spraydrift would potentially confound the results of the other plots, but washopefully somewhat minimized by the use of a 2 row border betweentreatments.

Within the 8.75 g/ha ai rimsulfuron treatment, the 4 GAT events werescored with significantly less spray damage compared to Jack and STS atboth 7 DAS (data not shown) and 14 DAS (data not shown). Among the 4 GATevents, there was no statistical difference noted at both rating times(data not shown). Examining the 35.0 g/ha ai rimsulfuron treatment, the4 GAT events were scored with significantly less spray damage comparedto Jack and STS at both 7 DAS (data not shown) and 14 DAS (data notshown). GAT7 Event EAFS 3560.4.3 was scored with less damage compared toGAT7 events EAFS 3559.2.1 and EAFS 3561.1.1 at both 7 DAS (data notshown) and 14 DAS (v).

After the 35.03 g/ha ai Synchrony treatment, the 4 GAT events werescored with significantly less spray damage compared to Jack and STS atboth 7 DAS (data not shown) and 14 DAS (data not shown). Among the 4 GATevents, there was no statistical difference noted at both 7 DAS (datanot shown) and 14 DAS (data not shown). In examining the 140.11 g/ha aiSynchrony treatment, the 4 GAT events were scored with significantlyless spray damage compared to Jack and STS at both 7 DAS (data notshown) and 14 DAS (data not shown). GAT7 events EAFS 3560.4.3 and EAFS3561.1.1 were scored with less damage compared to GAT7 events EAFS3559.2.1 and EAFS 3560.3.2 at 7 DAS (data not shown). At 14 DAS the140.11 g/ha ai Synchrony treatment, GAT7 events EAFS 3560.4.2, EAFS3559.2.1, and EAFS 3561.1.1 were rated with no damage, while EAFS3560.3.2 was scored similar to STS (data not shown).

In examining the 8.75 g/ha ai tribenuron treatment, the 4 GAT7 eventshad significantly less visual damage compared to STS and Jack at 7 DAS(data not shown) and 14 DAS (data not shown). The GAT7 events were notstatistically different at 7 DAS (data not shown), but Evens EAFS3560.4.3 and EAFS 3559.2.1 had significantly lower damage compared toEAFS 3560.3.2 at 14 DAS (data not shown). For the 35.0 g/ha aitribenuron treatment, the 4 GAT7 events were rated with significantlyless spray response compared to Jack and STS at 7 DAS (data not shown)and 14 DAS (data not shown). The 4 GAT events were not statisticallydifferent from each other at 7 DAS (data not shown), but the EAFS3560.3.2 was rated with significantly more spray damage than the other 3GAT7 events at 14 DAS (data not shown).

For the 3360.0 g/ha ai glyphosate and 3360.0 g/ha ai glyphosate at V3followed by 3360.0 g/ha ai glyphosate at R1 treatments, the Jack and STSvarieties were destroyed after 7 days, as expected (data not shown). The4 GAT7 events did not have any observed spray damage for the 3360.0 g/haai glyphosate treatment at 7 DAS (data not shown) and 14 DAS (data notshown). Minimal spray damage was recorded for the 4 GAT7 events at 7 DASfor the treatment (data not shown), and events EAFS 3560.4.3 and EAFS3559.2.1 were rated significantly better than EAFS 3560.3.2 at 14 DAS(data not shown).

Two tank-mix treatments of glyphosate plus a sulfonylurea herbicideprovided similar results at 7 DAS and 14 DAS. For the 3360.0 g/ha aiglyphosate plus 70.0 g/ha ai rimsulfuron treatment, the 4 GAT events hada similar herbicide response at 7 DAS of about 40% damage (data notshown), and approximately 35% damage at 14 DAS (data not shown). Less ofan overall crop response was observed for the 3360.0 g/ha ai glyphosateplus 35.0 g/ha ai tribenuron treatment, and the 4 GAT events were notstatistically different at 7 DAS (data not shown), and 14 DAS (data notshown).

The mean of visual ratings at 7 DAS and 14 DAS were graphed for the fourlead GAT events, Jack, and the STS line to allow visual interpretationof the data (data not shown). For the Rimsulfuron treatments, there wasa crop response observed with the 4 lead GAT events, with EAFS 3560.4.3having the most tolerance noted (data not shown). The response of theGAT7 events was significantly less than the STS and Jack controls forboth Rimsulfuron treatments at 7 DAS and 14 DAS (data not shown).

In examining the mean response scores to the 1× and 140.11 g/ha aiSynchrony treatments, there was no apparent damage for GAT7 events EAFS3560.4.3 and EAFS 3561.1.1, and minimal response of GAT 7 events EAFS3559.2.1 and EAFS 3560.3.2 (data not shown). All GAT events hadsignificantly less crop response compared to Jack and the STS line (datanot shown).

The four GAT7 events had significantly less crop response to 1× and 35.0g/ha ai tribenuron applications at 7 DAS and 14 DAS when compared toJack and STS (data not shown). Among the GAT7 events, EAFS 3560.4.3performed the best, while EAFS 3560.3.2 appeared to show the mostresponse overall (data not shown).

For the 3360.0 g/ha ai glyphosate application, there was no apparentcrop response for all 4 GAT7 events at 7 DAS and 14 DAS (data notshown). For the 3360.0 g/ha ai glyphosate at V3 followed by 3360.0 g/haai glyphosate at R1, there were no statistical differences noted amongthe four GAT7 events at 7 DAS, but EAFS 3560.4.3 and EAFS 3559.2.1appeared to have less response compared to EAFS 3561.1.1 and EAFS3560.3.2 at 14 DAS (data not shown).

Of the two tank mix treatments, the 70.0 g/ha ai rimsulfuron plus 3360.0g/ha ai glyphosate caused a higher level of crop damage responsecompared to the 35.0 g/ha ai tribenuron plus 3360.0 g/ha ai glyphosatetreatment (data not shown). Among the four lead GAT7 events, there wereno statistical differences observed for the visual ratings at 7 DAS and14 DAS (data not shown).

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method for controlling weeds in an area of cultivation comprisinga) planting the area with crop seeds or plants which comprise i) a firstpolynucleotide encoding a polypeptide that can confer tolerance toglyphosate operably linked to a promoter active in a plant; and, ii) asecond polynucleotide encoding an ALS inhibitor-tolerant polypeptideoperably linked to a promoter active in a plant; b) applying to thecrop, crop part, weed or area of cultivation thereof a combination ofherbicides comprising at least an effective amount of a first and asecond ALS inhibitor, wherein i) the effective amount of the combinationof herbicides controls weeds; ii) the effective amount of the first ALSinhibitor is not tolerated by the crop when applied alone when comparedto a control crop that has not been exposed to the first ALS inhibitor;and, iii) the effective amount of the second ALS inhibitor is sufficientto produce a safening effect, wherein said safening effect provides anincrease in crop tolerance upon the application of the first and thesecond ALS inhibitor when compared to the crop tolerance when the firstALS inhibitor is applied alone.
 2. The method of claim 1, wherein atleast said first or said second ALS inhibitor comprises a sulfonylureaor an imidazolinone.
 3. The method of claim 1, wherein said second ALSinhibitor comprises a sulfonylurea and the first ALS inhibitor comprisesan imidazolinone.
 4. The method of claim 3, wherein said sulfonylureacomprises rimsalfuron and said imidazolinone comprises imazethopyr. 5.The method of claim 1, wherein said combination of herbicides furthercomprises glyphosate.
 6. The method of claim 1, wherein said firstpolynucleotide encodes a glyphosate-N-acetyltransferase.
 7. The methodof claim 1, wherein said first polynucleotide encodes aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase or aglyphosate-tolerant glyphosate oxido-reductase.
 8. The method of claim1, wherein said ALS inhibitor-tolerant polypeptide comprises a mutatedacetolactate synthase polypeptide.
 9. The method of claim 8, whereinsaid mutated acetolactate synthase polypeptide comprises HRA.
 10. Themethod of claim 1, wherein said first and said second ALS inhibitorcomprise a homogeneous granule blend.
 11. The method claim 1, wherein atleast one of said first or said second polynucleotides is operablylinked to at least one copy of the enhancer sequence set forth in SEQ IDNO: 1, 72, 85, 88, or 89, or an enhancer sequence having at least 90%sequence identity to SEQ ID NO: 1, 72, 85, 88, or 89, wherein saidenhancer sequence modulates transcription.
 12. The method of claim 1,wherein said crop is a dicot.
 13. The method of claim 12, wherein saiddicot is soybean, canola, sunflower, cotton, alfalfa, an ornamental, afruit, a vegetable, a sugar beet, or an Arabidopsis.
 14. The method ofclaim 1, wherein said plant is a monocot.
 15. The method of claim 14,wherein said monocot is maize, wheat, rice, barley, sorghum, sugar cane,Switchgrass, or rye.
 16. The method of claim 1, wherein step (b) isperformed at least once prior to step (a).
 17. A method for controllingweeds in an area of cultivation comprising: a) evaluating environmentalconditions in an area of cultivation; b) selecting a combination ofherbicides comprising at least an effective amount of a first and asecond ALS inhibitor, wherein said effective amount of the combinationcontrols weeds, and the effective amount of the first ALS inhibitor isnot tolerated by the crop when applied alone when compared to a controlcrop that has been applied with the first ALS inhibitor, and theeffective amount of the second ALS inhibitor is sufficient to produce asafening effect, wherein said safening effect provides an increase incrop tolerance upon the application of the first and the second ALSinhibitor when compared to the crop tolerance when the first ALSinhibitor is applied alone; and, c) applying the combination ofherbicides to a crop, crop part, seed or an area of cultivation of saidcrop, wherein the crop comprises a plant having i) a firstpolynucleotide encoding a polypeptide that can confer tolerance toglyphosate operably linked to a promoter active in said plant; and, ii)a second polynucleotide encoding an ALS inhibitor-tolerant polypeptideoperably linked to a promoter active in said plant.
 18. The method ofclaim 17, wherein at least said first or said second ALS inhibitorcomprises a sulfonylurea or an imidazolinone.
 19. The method of claim17, wherein said second ALS inhibitor comprises a sulfonylurea and thefirst ALS inhibitor comprises an imidazolinone.
 20. The method of claim19, wherein said sulfonylurea comprises rimsalfuron and saidimidazolinone comprises imazethopyr.
 21. The method of claim 17, whereinsaid first polynucleotide encodes a glyphosate-N-acetyltransferase, andsaid ALS inhibitor-tolerant polypeptide comprises a mutated acetolactatesynthase polypeptide.